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UNIVERSITY OF CALGARY
Raphe Modulation of Circadian Phase
by
Glenn Yamakawa
A THESIS
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DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF PSYCHOLOGY
CALGARY, ALBERTA
SEPTEMBER, 2009
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1*1 Canada Abstract
The midbrain raphe nucleus provides a major input into the SCN, 50% of which contains serotonin. There is mixed evidence as to whether the serotonergic part of this projection is involved in non-photic phase shifting. In order to better characterize the non- serotonergic projections we conducted retrograde tract tracing. Approximately 30% of the projection contained VGLUT3, but not serotonin. To determine if these non- serotonergic projections were important for non-photic phase shifting, the MRN was stimulated in SCN 5-HT lesioned and sham control animals. Intact animals showed a phase advance to midday electrical stimulation of the raphe while the lesioned animals did not. Together, these results show the serotonergic raphe innervation of the SCN is important for non-photic phase shifting and that some of the non-serotonergic raphe to
SCN projections may contain glutamate.
11 Acknowledgements
I wish to thank my supervisor Dr. Michael Antle, for his patience, insight and allowing me the opportunity to conduct graduate studies in his lab. I wish to thank Dr. Ralph
Mistlberger for early opportunities to gain experience in the study of circadian rhythms. I also wish to thank Dr. Cam Teskey, Dr. Dave Glass, Dr. Brian Bland and Dr. John
Yeomans for their useful comments and guidance. I wish to thank Glenn Landry for being a mentor and the one who showed me what it took to be in neuroscience research. I also wish to thank my fellow graduate students in the Chronobiology lab, Andrew Brown from the Neural plasticity lab for stimulating discussion and Danica Whalley for technical assistance. Finally, I wish to thank the Natural Science and Engineering
Research Council and the Department of Psychology at the University of Calgary for funding this work.
m Dedication
I wish to dedicate this work to my family for their tremendous love and support. To my mom Cathy for her enthusiasm and strength, and my dad Dick for late night emails and teaching me to find something I am passionate about.
IV Table of Contents
Abstract ii Acknowledgements iii Dedication iv Table of Contents v List of Figures vii List of Abbreviations viii
CHAPTER ONE: GENERAL INTRODUCTION 1 1.1 Master Circadian Pacemaker 1 1.2 Non-Photic Phase Shifting 5 1.2.1 Molecular Effects of Non-Photic Stimuli 8 1.3 The Raphe Complex 10 1.4 Serotonin in the Circadian System 13 1.5 Electrical Stimulation Studies 15 1.6 Serotonin Agonists 15 1.7 Serotonin Antagonists 18 1.8 In Vitro Studies 19 1.9 Evidence Contrary to the Role of 5-HT 22 1.10 Current Studies 23 1.11 Vesicular Glutamate Transporters 24
CHAPTER TWO: RETROGRADE TRACT TRACING 26 2.1 Introduction 26 2.2 Animals and Apparatus 27 2.3 Experiment 1 27 2.3.1 Cholera Toxin p Subunit Ionotophoresis 27 2.3.2 SCN Cholera Toxin Immunocytochemistry 28 2.3.3 5-HT-VGLUT3-Cholera Toxin Immunocytochemistry 29 2.3.4 Analysis 30 2.4 Results 31 2.4.1 Median Raphe and Paramedian Raphe Labelling 33 2.4.2 Dorsal Raphe Labelling 34 2.5 Discussion 35 2.5.1 Retrograde Tract Tracing 35 2.5.2 The Role of VGLUT3 38 2.5.3 Methodological Considerations 42
CHAPTER THREE: MRN STIMULATION 43 3.1 Introduction 44 3.2 Experiment 2 45 3.3 Experiment 2B 46 3.4 Experimental Procedures 47 3.4.1 Perfusion and Immunocytochemistry 48 3.4.2 Analysis 49 3.5 Results 50
v 3.5.1 Behavior during Stimulation 50 3.5.2 Histology 51 3.6 Experiment 2 51 3.6.1 Control Stimulation Procedures 51 3.6.2 Stimulation of the Median Raphe 52 3.7 Experiment 2B 53 3.7.1 Stimulation of the Median Raphe 53 3.8 Discussion 54 3.8.1 What is the Zeitgeber? 54 3.8.2 Circling Behavior 57 3.9 Experimental Design Issues 59
CHAPTER FOUR: GENERAL DISCUSSION 61 4.1 Conclusions 61 4.2 Future Directions 63 4.3 Clinical Importance/Relevance 64
REFERENCES 68
VI List of Figures
Figure 2.1 Photomicrograph cases of iontophoresis into the SCN 94
Figure 2.2 Representative triple label histology and mean distribution of cell types retrogradely labelled in the MRN 95
Figure 2.3 Photomicrographs displaying representative examples of retrogradely labelled cell types in the MRN 97
Figure 2.4 Iontophoresis site in the SCN and retrograde labelling in the DRN for one case 98
Figure 2.5 Mean distribution of cell types retrogradely labelling in the DRN 99
Figure 3.1 5-HT labelled histology showing MRN electrode tip placements 101
Figure 3.2 5-HT labelled sections showing intact and lesioned serotonergic input
into the SCN 102
Figure 3.3 Mean phase shifts to control and MRN stimulation for experiment 2 .... 103
Figure 3.4 Actograms displaying an animal that received a control manipulation and another that received stimulation of the MRN 105 Figure 3.5 Mean phase shifts to MRN stimulation following cannula injection of vehicle, or 5,7-DHT 106 Figure 3.6 Mean phase shifts to MRN stimulation following vehicle of 5,7-DHT injection for experiment 2B 107
Figure 3.7 Actogram displaying an animal that received a complete lesion of the serotonergic input into the SCN and subsequent stimulation of the MRN 108
vn List of Abbreviations
Measurement Terms: Hz hertz Ms millisecond °C degrees Celsius M molar Hrs hours Min minutes Cm centimeters um micrometer uA microampere ml milliliter
Other Terms: 5-HT 5-hydroxytryptamine, or serotonin 5-HTR 5-HT transporter 5,7-DHT 5,7-dihydroxytryptamine 8-OH-DPAT 8-hydroxy- 2-(di-n- propylamino) tetralin ABC avidin-biotin complex ANOVA analysis of variance AP anterior/posterior cAMP cyclic adenosine monophosphate CT circadian time CTb cholera toxin beta subunit Cy 2,3,5 Cyanine 2,3, or 5 DAB diaminobenzidine DD constant dark DRN dorsal raphe nucleus DV dorsal/ventral GABA gamma-Aminobutyric acid GHT geniculohypothalamic tract ICC immunocytochemistry IGL intergeniculate leaflet LD light/dark LL constant light ML medial/lateral MRN median raphe nucleus mRNA messenger ribonucleic acid NAAG n-acetyl-aspartyl-glutamate NPY neuropeptide Y PBS phosphate buffered saline Perl period 1 p-ERK phosphorylated extracellular signal responsive kinase PRC phase response curve RHT retinohypothalamic tract
Vlll SCN suprachiasmatic nucleus TGF transcription growth factor TH tyrosine hydroxylase VGLUT vesicular glutamate transporter ZT zeitgeber time
IX 1
Chapter One: General Introduction
1.1 Master Circadian Pacemaker In today's society of around the clock productivity, trans-meridian travel and shift work it is important to examine ways to function at our best even when our bodies tell us otherwise. Many of the recorded disasters such as the Exxon Valdez, Chernobyl, and
Three Mile Island all occurred at times when cognitive and physical functioning was reduced as those involved were on the job during times when they normally would be resting (Folkard and Tucker, 2003). The phenomenon of jet lag is a widespread condition affecting thousands of travelers per year. Jet lag occurs when there is desynchrony between the sleep/wake cycle and the environment that can result in a wide range of symptoms including drowsiness, irritability and gastrointestinal problems (Revell and
Eastman, 2005). A similar desynchrony occurs when paramedics, police officers and fire fighters are often called upon to perform complex tasks at night, or in the early morning when they are not operating at their peak efficiency. Recently, it was reported that animals unable to synchronize to the daily light/dark (LD) cycle suffered from extensive cardiovascular and renal damage with dramatically shortened life spans (Martino et al.,
2008). There is also accumulating evidence that exposure to light at night in shift workers may lead to an increased risk of cancer (Hansen, 2001). Modern chronobiology has sought to understand how the circadian system functions and develop interventions for dealing with some of these issues.
Some form of biological time keeping system is highly preserved from single cell organisms to mammals. Synchronizing biological processes to the daily LD cycle is 2 crucial in an ecological and evolutionary sense. It has recently been proposed that early metazoans evolved means to avoid damaging ultraviolet radiation from the sun by descending deeper in the ocean when the sun was up (Gehring and Roshbash, 2002).
These were probably the first form of a circadian timing system, as it would have required an endogenous daily rhythm of activity that could allow the organism to anticipate sunrise by descending and return to the surface at nightfall. Cyanobacteria strains with circadian periods similar to the LD cycle were able to outcompete mutant strains with circadian periods different from the LD cycle indicating circadian clocks may also enhance reproductive fitness (Ouyang et al., 1998). The term circadian comes from the Latin for "circa", or about and diem "day." A circadian rhythm then, is a biological process that takes about a day to oscillate. There is some variability both within and between species in how long it takes for the oscillation to occur. Exactly how long it takes for these processes to oscillate under constant conditions is referred to as the organism's circadian period.
Emerging evidence of a master circadian clock in mammals started research in the field of biological rhythms. Early work to elucidate the biological underpinnings of circadian rhythms began with the observations that the nocturnal rhythms of corticosterone release, locomotor activity and drinking behaviour were lost following suprachiasmatic nucleus (SCN) ablation (Moore and Eichler, 1972; Stephan and Zucker,
1972; Satinoff and Prosser, 1988). The SCN is a highly heterogenous bilateral distribution of approximately 20000 cells located ventral to the third ventricle of the anterior hypothalamus. Glucose utilization studies revealed the SCN of female rats were more active during the light period than the dark, and this rhythm also persisted in the 3 absence of light cues (Schwartz and Gainer, 1977). When the SCN of enucleated animals was separated from the rest of the brain by a knife cuts in vivo, it still retained rhythmicity of electrical activity while other brain structures did not (Inouye and
Kawamura, 1979). Similarly, some SCN cells retained their spontaneous rhythmic electrical firing in vivo and in vitro (Groos and Hendriks, 1979). Mice bred to have graded underdevelopment of the SCN and optic systems were unable to synchronize, or entrain to a 12:12 (12 hours of light followed by 12 hours of dark) LD cycle and showed free running or arrhythmic wheel running rhythms depending on the region of cell loss in the SCN (Scheuch et al., 1982). It was concluded that the SCN functions to produce an endogenous rhythm of activity close to 24 hours and to entrain, or synchronize, an organism to certain stimuli (Morin, 1999).
Perhaps the most indisputable evidence that the SCN functioned as the master pacemaker came after the discovery of an autosomal mutation in a hamster that conveyed a natural period of circadian rhythms (tau) that was 22 hours in the heterozygous mutation and around 20 hours in the homozygous case. This short tau fell well short of the established average tau for normal hamsters of approximately 24.1 hours and was therefore named the tau mutant hamster (Ralph and Menaker, 1988). Arrhythmic, SCN lesioned animals that received intact SCN tissue transplants into the third ventricle had circadian rhythms restored to the donor's natural period (Ralph et al., 1990). Thus, if a hamster with a normal tau of 24.1 hours received a SCN lesion and subsequent transplant from a tau mutant animal, the animal adopted the donor's abnormally short period of 20-
22 hours. It was unequivocally concluded that circadian rhythmicity within the mammalian nervous system is generated by the SCN of the ventral hypothalamus but 4 discovering how the biological clock worked was only just beginning (Moore et al.,
2002).
It has been known for some time that the light exposure has a great influence on behaviour and physiology. Light has been found to be the dominant zeitgeber "time giver" (stimulus that sets the clock) for the SCN that is capable of entraining, or shifting the endogenously rhythmic clock. Photic information reaches the SCN via a direct pathway along the retinohypothalamic tract (RHT). Exposure to light in the early subjective night results in phase delays to the clock with phase advances occurring in the late subjective night (Pittendrigh and Daan, 1976; Foster and Kreitzman, 2004). A phase delay refers to an animal's biological rhythms being reset, or delayed to a later time on subsequent days following the light stimulus, while the phase advance refers to a shift in the biological rhythms to an earlier time following the light stimulus. Exposure to light in the subjective day does not result in any phase shifts and is known as the dead zone. A plot of this pattern of phase shifts is known as the photic phase response curve (PRC).
There are also numerous stimuli capable of shifting the clock in a manner inconsistent with the photic PRC. Collectively, these are referred to as non-photic stimuli.
Examples of non-photic stimuli include access to a novel running wheel, dark pulses,
(exposure to darkness when an animal is being kept in constant light) saline injection, benzodiazepines, sleep deprivation, and social stimuli (Yannielli and Harrington, 2004).
Exposure to a non-photic stimulus in the mid-subjective day results in large phase advances of the circadian activity rhythm while exposure in the early part of the night results in smaller phase delays. This pattern of advances and delays is referred to as the non-photic PRC. 1.2 Non-Photic Phase Shifting Some of the first non-photic manipulations studied were dark pulses. Animals maintained in constant light (LL) are phase shifted by dark exposure (known as dark pulses) in a non-photic phase dependent manner. Hamsters exposed to dark pulses in mid-subjective day often begin wheel running vigorously. Moderate (3 hr) and long (6-9 hr) dark pulses resulted in elevated locomotor activity that was highly correlated with the magnitude of phase shift (Canal and Piggins, 2005). Hamsters in LL but confined to a novel wheel in the absence of a dark pulse showed phase shifts comparable in magnitude to dark pulses indicating "dark" may not be the zeitgeber and instead it may be the wheel running (Reebs et al., 1989). However, animals given a midday 4-hour dark pulse in the absence of a running wheel also showed significant phase advances (Mendoza et al.,
2004).
Dark pulses result in significant down regulation of phosphorylated extracellular signal responsive kinase (p-ERK) and FOS in the SCN at mid-subjective day (Coogan and Piggins, 2005). Dark pulses at circadian time 4 (CT4; By convention, CT12 is defined as activity onset for a nocturnal animal) and CT8 significantly downregulated
Perl and Perl but only Perl was down regulated at CT12 (Mendoza et al., 2004). There is also a down regulation of transforming growth factor-a (Tgf-a) in the SCN at CT4 and
CT12 dark pulse exposures (Mendoza et al., 2007). Interestingly enough, TGF-a has been found to inhibit locomotion during the rest period of mice through epidermal growth factor receptor signaling (Kramer et al., 2001). It cannot be said whether TGF-a is downregulated due to the non-photic stimulus, or is directly involved in eliciting the non- photic phase shift. 6
Ideas and arguments over what exact zeitgeber of a dark pulse was resulting in the non-photic phase shift gave rise to research into other stimuli. For example, it could have been the "dark" that shifted the clock, the "wheel running," the "sleep deprivation" during the rest period, or any other number of specific stimuli associated with the dark pulse that shifted the clock. In general, true non-photic stimuli involve some form of sustained arousal. Early work in non-photic phase shifting described re-entrainment to an
8-hour advance of the LD cycle faster in animals confined to a novel running wheel at mid-subjective day (Mrosovsky and Salmon, 1987). In order to determine if the novel wheel access was actually resulting in a phase shift of the clock, another study was conducted. Animals placed in constant dark (DD) after novel wheel access became active at a time several hours advanced from the previous LD cycle (Mrosovsky, 1989). This indicates that the novel wheel access was resulting in a shift in the circadian rhythm of activity and not merely forcing the animal to be active at a different time. Novel wheel exposure during mid-subjective day in hamsters has been shown to result in phase advances in excess of three hours (Bobrzynska et al., 1996a). Maximal phase shifts occur with novel wheel access that is close to 3 hours long (Reebs and Mrosvosky, 1989). If kept in LD without access to a running wheel and then transferred to DD with access to a wheel, phase shifts as long as 15 hours have been reported (Gannon and Rea, 1995). The magnitude of phase shift can be reliably predicted based on how much the hamster runs in the wheel during these manipulations. Bilateral electrolytic lesions of the intergeniculate leaflet (IGL) of the thalamus blocked or attenuated phase shifts to novel wheel access, however, also reduced overall activity making this finding difficult to interpret (Janik and Mrosovsky, 1994). Neuropeptide Y (NPY) administration at 7 zeitgeber time 4 (ZT4; By convention ZT12 is specified as the time when the lights turn off in an LD cycle) phase advanced hamsters independently of locomotor activity, and anti-sera against NPY greatly reduced shifts to novel wheel access (Biello et al., 1994;
Wickland and Turek, 1994). These studies suggest that NPY is involved in non-photic phase shifting.
Sleep deprivation by gentle handling has been suggested as a way of dissociating arousal from excess locomotor activity in order to further elucidate the non-photic zeitgeber. A 3-hour sleep deprivation by gentle handling at mid-subjective day under dim red light induces phase advances of circadian rhythms of wheel running by around 2 hours (Grossman et al., 2000). During a sleep deprivation by gentle handling, the total distance traveled by the animal was 0.08 km, compared to 2.5 km for typical responders in a novel wheel access manipulation (Antle and Mistlberger, 2000). Since the magnitude of phase shift in a sleep deprivation is comparable to that of novel wheel access, motor activity is sufficient, but not necessary to produce a non-photic phase shift. It could be the case that all non-photic stimuli have sustained arousal causing sleep deprivation as a common zeitgeber that is shifting the clock. Perhaps the organism lacks sleep so the clock readjusts the active period to an earlier time in order to rest at an earlier time on subsequent days.
Melatonin is known to phase advance rhythms in late subjective day, but have no effect at any other circadian time. Manual injections of melatonin or saline were observed to be highly arousing to hamsters as they often began wheel running immediately following the manipulation. When melatonin and saline injections were given remotely through a subcutaneous implanted cannula, however, no phase shifts to these substances 8 occurred (Hastings et al., 1992). Again, the evidence points towards some kind of specific arousal being necessary for non-photic phase shifting.
There are numerous ways of arousing an animal including sexual stimuli, opportunity to hoard, social cues and cage changing (reviewed in Mrosovsky, 1996).
These stimuli seem to be consistent with the non-photic PRC, and require at least an hour or more in order to be effective. A recent c-fos expression study revealed novel wheel access and sleep deprivation, along with stress by physical restraint which does not shift the clock, increased Fos in the IGL and hypocretin arousal promoting neurons.
Surprisingly, arousing doses of caffeine and modafinil failed to shift the clock and also failed to activate these hypocretin neurons despite the fact that the animals remained awake and highly aroused throughout the effects of the drugs (Webb et al., 2008).
1.2.1 Molecular Effects of Non-Photic Stimuli
Although the underlying biological mechanism of these non-photic phase shifts is not well known, these stimuli have been found to alter gene expression in some important circadian structures. The immediate early gene c-fos increases in the IGL and pretectum with a non-photic stimulus, but Fos levels decrease or remain unchanged in the SCN
(Janik and Mrosovsky, 1992). Retinal afferents also project to the IGL of the thalamus.
From the IGL, photic information may be integrated with behavioural feedback and indirectly reach the SCN along the NPYergic geniculohypothalamic tract (GHT) (Card and Moore, 1989). There were no changes in another immediate early gene FosB in response to non-photic manipulation of any area compared to home cage controls
(Mikkelsen et al., 1998). Another study found SCN and IGL Fos immunoreactivity 9 increased in response to non-photic manipulations (Janik et al., 1995). On the contrary, it has also been reported that almost none of the IGL projections to the SCN showed Fos induction to novel wheel access (Muscat and Morin, 2006). Midday non-photic manipulations also resulted in lower levels of SCN clock gene Periodl {Perl) messenger ribonucleic acid (mRNA) at ZT7 and lowered Perl mRNA at ZT9 (Yanielli et al., 2002).
Suppression of Perl expression in the SCN using antisense oligodeoxynucleotides results in large phase advances during the day and small delays during subjective night (Hamada et al., 2004). Non-photic shifts have also been found to decrease levels of p-ERK and pharmacologically blocking phosphorylation of this kinase did not elicit non-photic phase shifts (Antle et al., 2008). P-ERK has already been shown to have rhythmic expression in the SCN and be involved in photic phase shifting (Coogan and Piggins, 2003). These results suggest that p-ERK might play a role in non-photic shifting as well.
Similar to light, non-photic stimuli must have a way of contacting the SCN in order to deliver the phase resetting information. There is strong evidence that NPY from the IGL of the thalamus acts at the SCN to produce non-photic phase shifts (reviewed by
Mrosovsky, 1996). Non-photic manipulations are thought to reach the SCN via the IGL and GHT (reviewed by Mrosovsky, 1996). Central injections of NPY into the SCN of hamsters results in phase shifts consistent with the non-photic PRC (Albers and Ferris,
1984). Serotonin, or 5-hydroxytryptamine (5-HT) is also thought to play a role in non- photic phase shifting, but the evidence is mixed. Some studies report a role for 5-HT in non-photic phase shifting, but other studies challenge that notion.
One of the major inputs into the SCN contains 5-HT where it plays a role in the regulation of circadian rhythmicity. There is ample evidence of the involvement of 5-HT 10 in the modulation of photic shifts in the SCN (e.g., Ying and Rusak, 1994). There are also some studies that suggest 5-HT release at the SCN is involved in non-photic phase shifting. During midday novel wheel access and sleep deprivations, there is a marked increase in SCN and IGL 5-HT levels (Dudley et al., 1998; Grossman et al., 2004).
Recently, 2-hour dark pulses were shown to result in an increase of SCN 5-HT and Fos levels in the raphe nuclei while these dark pulse phase shifts were potentiated by the 5-
HTIA/7 agonist 8-Hydroxy- 2-di-n- propylamino tetralin (8-OH-DPAT) (Mendoza et al.,
2008). In the Djungarian hamster, a 4-hour sleep deprivation by gentle handling was shown to increase 5-HT turnover in the hypothalamus (Asikainen et al., 1995).
1.3 The Raphe Complex
The raphe nuclei are the primary source of 5-HT in the mammalian brain. There is an ascending system of projections coming from the rostral raphe group consisting mainly of the median and dorsal raphe nuclei (MRN and DRN) and a descending system of projections consisting primarily of the nuclei raphe magnus, obscurus and pallidus
(Jacobs et al., 2002). The rostral ascending raphe system projects widely to almost every forebrain region and thus has been implicated in a variety of functions such as mood, feeding, sexual behaviors, sleep, thermoregulation and pain reception (Verge and Calas,
2000). Serotonergic cells are active when muscle tone is high such as during wake, and become suppressed or inactive and when there is little or no muscle tone, such as during sleep (Jacobs and Azmitia, 1992).
Using retrograde and anterograde tracing in Syrian hamsters, the DRN was found to send serotonergic fibers to the IGL, and the MRN sent a substantial projection to the 11
SCN, half of which was serotonergic (Meyer-Bernstein and Morin, 1996). Anterograde tracing studies have also been conducted. It was confirmed that the DRN and MRN generally projected to the same areas with denser projections coming from the larger
DRN. The DRN heavily innervated most of the lateral and dorsal regions of the anterior hypothalamus, however not the arcuate, ventromedial hypothalamus or SCN whereas the
MRN exclusively sent modest projections to the SCN (Morin and Meyer-Bernstein,
1999). Following iontophoresis of anterograde tracer into the MRN, dense staining was found in the SCN roughly corresponding to the ventromedial fiber plexus of serotonin input (Meyer-Bernstein and Morin, 1996). A later follow-up anterograde tracing study confirmed findings of an exclusive MRN input into the SCN and a DRN projection to the
IGL with the exception of one case that appeared to show a DRN to SCN projection
(Morin and Meyer-Bernstein, 1999). A retrograde labelling study from the SCN confirmed and extended observations that the MRN, raphe magnus and raphe obscurus all project to the SCN and only about half those fibers from the MRN were 5-HT immunoreactive in the rat (Hay-Schmidt et al., 2003). Although the evidence suggests exclusive serotonergic innervation of the SCN, there are some findings to the contrary.
Anterograde tracing in the golden hamster revealed a dense median raphe innervation of the ventrolateral SCN; however several fibers in the dorsal tip of the SCN were also reported following DRN tracing (Leander et al., 1998). Similarly, an older study found equal numbers of bilaterally distributed neurons in the DRN and MRN of the golden hamster projected to the SCN (Pickard, 1982). In the rat, some retrograde labelling has revealed a partially serotonergic projection from the DRN to the SCN while anterograde 12 tracing from the eyes showed evidence for a direct retina to DRN projection (Kawano et al., 1996).
There is also some evidence of a reciprocal projection between the DRN and
MRN. Retrograde tract tracing has revealed the dorsal raphe projects to the median and paramedian raphe and that there is another partially serotonergic connection from the
MRN to the DRN (Tischler and Morin, 2003). Additional raphe afferents include DRN projections to the basal ganglia and the MRN connections to limbic, parietal and occipital structures (Jacobs and Azmitia 1992).
Both the DRN and MRN as whole nuclei exhibit endogenous rhythms of slow, highly regular discharge patterns that persist (albeit with a higher amplitude) with transaction from the forebrain (reviewed by Jacobs and Azmitia, 1992). Both subsets of neurons are relatively undisturbed by sensory, or environmental stimulation and activity in the DRN seems to be related to muscle tone (Jacobs and Azmitia, 1992).
Electrophysiological studies have found the MRN contains a subset of non-5-HT neurons that differ substantially from the non-serotonergic neurons in the DRN (Beck et al. 2004). This suggests that the MRN contains a unique subset of neuronal projections that have not yet been identified. Electrophysiologically, it is believed that the non-5-HT neurons in the DRN are serotonergic early in development but the non-5-HT neurons in the MRN show greatly different electrophysiological profiles for this to be the case.
These non-5-HT DRN neurons are very similar to the 5-HT neurons on whole cell recording measures; however non-5-HT MRN neurons differ substantially from serotonergic cells (Beck et al., 2004). 13
1.4 Serotonin in the Circadian System
Complete 5,7-dihydroxytryptamine (5,7- DHT) neurotoxic lesions of the MRN serotonergic cells results in a large advance in hamster wheel running activity onset, a delay in offset and increase in the active period (alpha) versus control and serotonin SCN lesioned animals (Meyer-Bernstein et al., 1997). This indicates at least partial 5-HT modulation of important circadian parameters such as offset and alpha.
Levels of tryptophan hydroxylase, the rate limiting enzyme in 5-HT synthesis were shown to have a circadian rhythm of expression with a peak towards dark onset in the MRN and DRN that persisted in continual darkness (Barassin et al., 2002). In a 12:12
LD cycle, 5-HT expression in the SCN of rats showed a diurnal rhythmicity of expression with a peak around ZT4, when transferred to DD the peak shifted to CT16 and in LL two peaks of expression appeared at CT4 and CT20 (Cagampang and Inouye, 1994). Using in vivo microdialysis, hamster SCN 5-HT levels were found to show an endogenous rhythm with a sharp increase at the light/dark transition followed by a steady decline; this release was more readily suppressed by autoreceptor agonism in the dark phase (Dudley et al.,
1998). The numerous effects of serotonin are complicated by the fact of its widespread projections to early every part of the brain where it can exert a tonic modulatory influence. At least 14 different subtypes of 5-HT receptors have been identified. Due to the wide range of receptor subtypes, 5-HT can mediate an extensive array of physiological effects (for a review see Barnes and Sharp, 1999).
Earlier in situ hybridization studies in the SCN revealed the heavy presence of 5-
HTic receptors with less 5-HTIB signal as well as scattered 5-HTIA and 5-HT2 (Roca et al., 1993). The 5-HT iA receptor can be localized as a pre-synaptic autoreceptor or a post- 14 synaptic binding site. Later immunocytochemical localization attempts determined 5-
HT2C receptors were densely distributed in the ventral regions of the SCN, and there was a uniform distribution of 5-HT2A fibers throughout the SCN and optic chiasm (Moyer and
Kennaway, 1999). In this same study, no staining was found for 5-HT7 receptors. A later study revealed there to be a strong evidence for the presence of 5-HT7 receptors in the
SCN through in situ hybridization, immunocytochemistry and stimulation of Fos expression (Neumaier et al., 2001).
Using a specific antibody raised against 5-HT5A receptors in the hamster, it was found that the DRN, MRN, IGL, and SCN all contained these receptors and it was suggested that they may act as an additional presynaptic autoreceptor along with the 5-
HTIA to mediate some of the phase shifting effects of 5-HT (Duncan et al., 2000).
Similarly, a high degree of 5-HTSA receptor immunoreactivity was found in the rat SCN using gel electrophoresis (Oliver et al., 2000). It has been argued that the relative unspecificity of pharmacological agents does not allow unambiguous differentiation between the 5-HTSA and the 5-HT7 receptors that are also present in the SCN though
(Gannon, 2001). The 5-HT5A and 5-HT7 receptors may both play important roles in mediating circadian rhythms.
The glycoprotein 5-HT transporter (5-HTR) has been localized on neurons in both the SCN and IGL (Amir et al., 1998). 5-HTR is responsible for re-uptake of synaptic 5-
HT. Within the SCN, 5-HTR was found in close proximity to the retinorecipient cells of the SCN. In hamsters, 5-HTR is distributed in a mainly overlapping pattern to the dense
5-HT fibers in the ventromedial area of the SCN (Legutko and Gannon, 2001). 15
1.5 Electrical Stimulation Studies
Crucial in demonstrating the role of 5-HT in the SCN during non-photic phase shifting is electrical stimulation of the raphe complex. Small phase advances of hamster wheel running are seen with stimulation of either the MRN or DRN during the mid- subjective day with small phase delays occurring in the early subjective night in LL
(Meyer-Bernstein and Morin, 1999). Interestingly, it was also reported in this study that an increase in locomotor activity accompanied raphe stimulation which is the opposite of what is expected given studies in the rat that report activity suppression following raphe activation (e.g., Wirtshafter and McWilliams, 1987). Stimulation of the MRN or DRN also results in an increase of 5-HT release in the SCN. MRN stimulation or microinjection of the 5-HTiA autoreceptor antagonist WAY 100635 both increase 5-HT release in the SCN while injections of 8-OH-DPAT into the MRN inhibited this effect presumably through 5-HT autoreceptor mediated inhibition (Dudley et al., 1999). In a study using DRN stimulation at mid-subjective day during DD, phase advances as long as
1.3 hours were reported (Glass et al., 2000). These phase shifts were thought to act through a multisynaptic pathway from the DRN to the MRN as they were suppressed by
5-HT7 antagonism in the DRN or MRN (Glass et al., 2003). This does not rule out DRN activation of the IGL but 8-OH-DPAT injections into the IGL fail to shift the clock, ruling out the 5-HTiA/7 receptor mediated pathways (Mintz et al., 1997).
1.6 Serotonin Agonists
Numerous pharmacological substances have been utilized in an attempt to elucidate the precise mode of action of 5-HT on the SCN. Injection of the gamma amino- butyric acid (GABA)A agonist muscimol into the DRN attenuates phase shifts to novel wheels in responders (Glass et al., 2003). This indicates at least a partial GABA modulation of 5-HT in the raphe and the possibility that 5-HT may partially mediate phase shifts to novel wheel access. In Syrian hamsters, 8-OH-DPAT administered systemically produced large phase advances at midday that were blocked by neurotoxic
5,7-DHT lesions of 5-HT afferents to the SCN (Schuhler et al. 1998; Ehlen et al., 2001).
Phase advances are also seen with CT6 bilateral microinjection of 8-OH-DPAT into the
SCN or IGL of hamsters in DD (Challet et al., 1998). Midday phase advances were also found with 3 hr timed perfusions of 5-HT or 8-OH-DPAT into the SCN for durations more equivalent to non-photic stimuli (Ehlen et al., 2001). The shifting properties of 8-
OH-DPAT are significantly enhanced by pretreatment with the 5-HT synthesis inhibitor p-chlorophenylalanine and greatly attenuated by 5-HT7 antagonists (Ehlen et al., 2001).
The phase shifting effects of 8-OH-DPAT and sleep deprivation are also significantly potentiated by prior exposure to LL that is not due to the upregulation of receptors in the
SCN of raphe (Knoch et al., 2004; Duncan et al., 2005). It is suspected the effects of LL are due to modification of the light responsive clock gene expression in the SCN. 8-OH-
DPAT was found to inhibit expression of Perl and Per2 in the SCN during the mid- subjective day, but not in the early day, or subjective night (Horikawa et al., 2000).
A potential confound in the 8-OH-DPAT studies was controlling for excessive motor activity during drug administration of the drug in order to be certain 5-HT agonism was an effective non-photic zeitgeber. 8-OH-DPAT phase shifts were no different if the animal was confined in a nest box and prevented from being active following the 17 injection (Bobrzynska et al, 1996a). However, when preventing activity following injection of the benzodiazepine triazolam, no phase shifts were found.
CT6 central injection of 8-OH-DPAT into the ventricles of rats produced highly variable (0-120 min) phase advances with or without access to a running wheel but had no effect at CT18 (Edgar et al., 1993). In the same study, the general 5-HT agonist quipazine produced 15-150 min advances at CT6 with occasional delays at CT18 administrations. Quipazine also sporadically produced a shortening of tau in the higher dose range. Interestingly enough, a study using three different inbred strains of rats showed subcutaneous injections of quipazine caused delays, advances and Fos expression in the SCN following a photic-like phase response curve (Kohler et al. 1999). This indicates that there may be substantial species differences in response to serotonin.
Animals receiving IGL lesions or serotonergic ablation in the SCN still phase shifted to systemic quipazine however phase shifts were eliminated in those receiving bilateral enucleation (Graff et al., 2005). With bilateral electrolytic or serotonergic lesions of the
IGL, midday phase advances to both 8-OH-DPAT and triazolam are abolished (Schuhler et al., 1999). The selective 5-HT2C receptor agonists DOI and RO-60-0175 have also been evaluated for their ability to alter clock gene expression in the SCN and produce phase shifts. Systemic administration of either drug at ZT16 in rats induced c-fos, Perl and Per2 expression in the SCN at the early night (Varcoe et al., 2003). This indicates 5-
HT2C involvement in circadian rhythmicity.
Melatonin has been shown to inhibit serotonergic phase advances in vitro presumably though reducing the availability of the second messenger cyclic adenosine monophosphate (cAMP) (Prosser, 1999). Administration of melatonin in conjunction 18 with novel wheel access failed to inhibit shifts, however (Antle et al., 2002). An in depth analysis of several serotonin agonists was conducted on mice using hamsters as a comparison. Mice failed to shift to systemic administration of the 5-HT]A/7 agonist 8-
OH-DPAT or quipazine across the wide range of circadian times. Hamsters, on the other hand, showed moderate phase advances to systemic 8-OH-DPAT at CT 8 but did not shift to quipazine (Antle et al., 2003). However, recent work suggests that systemic administration of 8-OH-DPAT in mice results in phase advances at CT6, a time not tested in the previous study (Horikawa and Shibata, 2004; Gardani and Biello, 2008). 8-OH-
DPAT does not appear to have a phase shifting effect in mice at other circadian times.
1.7 Serotonin Antagonists
Gannon et al., (2003) evaluated intraperitoneal administration of mixed 5-HTIA agonist/antagonist BMY 7378 in Syrian hamsters. BMY 7378 is a functional antagonist as it acts as an agonist at the presynaptic 5-HTiA autoreceptor and an antagonist at the postsynaptic 5-HT]A receptor. They found phase advances of up to an hour when administered by itself during the night. A similar study administering BMY 7378 at CT19 showed phase advances of 1-2 hours and advances close to an hour when co-administered with the NPY antagonist CP-760, 542 (Lall and Harrington, 2006). WAY 100635 has been evaluated as a pharmacological 1A receptor antagonist and has been suggested to be
'silent' or have little to no other effects (Forster et al., 1995). It was shown that 5-HT2A, or 5-HT2c agonism or antagonism was unable to produce phase shifts in hamsters
(Gannon and Millan, 2006). Metergoline is a known as a 5-HT1/2/7 antagonist.
Administration of metergoline following dark pulses in constant lighting conditions 19 attenuated phase shifts in mice however fails to attenuate phase advances to novel wheel access in hamsters (Bartoszewicz and Barbacka-Surowiak, 2007; Antle et al., 1998).
Ritanserin, a 5-HT7 receptor antagonist and has been shown to dose dependently inhibit phase advances to 8-OH-DPAT in rats but also, had no effect on wheel confinement in hamsters (Sprouse et al., 2004; Antle et al., 1998).
1.8 In Vitro Studies
In vitro preparations of rat SCN slices show a persistent rhythm of electrical activity for around three days with peak discharge rates occurring during the day (Yu et al., 2001). Midday hour-long application of cAMP analogs causes 4-6 hr phase advances in electrical activity of SCN neurons with no effects at other time points (Gillette and
Prosser, 1988; Prosser and Gillete, 1989). cAMP levels are normally high during the late day and late night (Prosser and Gillette, 1991). This suggests the neurotransmitter responsible for inducing non-photic phase advances during the day stimulates the accumulation of the second messenger cAMP. Activation of the cyclic guanosine monophosphate pathway appears to be important at night (Prosser et al., 1989). A good candidate receptor mediating stimulation of cAMP accumulation and subsequent phase shifts through activation of adenylyl cyclase is the 5-HT7 receptor (described in
Lovenberg et al., 1993). Therefore the 5-HT7 receptor has been implicated in vivo and in vitro as well.
CT7 microdrop application of 5-HT, 8-OH-DPAT, or 5-carboxaminotryptamine to rat SCN slices resulted in very large (greater than 6 hrs) phase advances of electrical activity with no observable effects occurring during the subjective night (Medanic and 20
Gillette, 1992; Shibata et al., 1992). In a series of experiments using in vitro bath application, administration of quipazine to rat SCN slices dose dependently advanced cell firing when administered in the day but also delayed cell firing in the night similar to non-photic stimuli (Prosser et al., 1990). In a follow up study, it was confirmed that in vitro administration of quipazine mimicked the effects of 5-HT and was completely blocked by pre-treatment with metergoline, once again suggesting the involvement of the
5-HT7 receptor (Prosser et al., 1993). In sum then, 5-HT and the relatively non-specific 5-
HT agonist quipazine were able to produce advances in the day and delays at night and it was suspected that 5-HT7 receptors mediated these effects.
8-OH-DPAT and another 5HTiA agonist buspirone, both only induced phase advances during the day that was blocked by NAN-190 a 5-HTiA antagonist. It was therefore concluded that the phase advancing effects of 5-HT was at the 1A receptor during the day (Prosser et al., 1993). Since 5-HTiA antagonism was unable to block the
5-HT and buspirone induced phase delays though, the authors concluded that early night phase delays likely acted through a different receptor. It is important to note that in vitro bath application may have a larger effect as 5-HT receptors are seen throughout the hypothalamus and the bath would likely activate targets outside the SCN as well.
Quipazine and 8-OH-DPAT advances can be blocked by cAMP inhibition and K+ channel blockers suggesting a role for cAMP-dependent protein kinase and potassium channels in serotonergic phase shifts (Prosser et al., 1994a). Quipazine application has also been shown to selectively decrease the normally high levels of c-fos mRNA in the dorsomedial SCN during the day but have no effect at night also indicating a differential mode of action for phase delays (Prosser et al., 1994b). Together, these results suggest the 5-HT7 and 5HTIA receptors are important for non-photic shifting.
In a study using rat SCN slices, 5-HT and 8-OH-DPAT produced large phase advances at CT6 that were dose dependently blocked by AMPA, a glutamate analog, but
NMDA had no effect unless it was co-applied with 8-OH-DPAT (Prosser, 2001). 8-OH-
DPAT induces the greatest shifts at ZT6 whereas application of NPY only phase advanced rat SCN slices at ZT10 and could inhibit 8-OH-DPAT shifts, but not vice versa
(Prosser, 1998). In a study using whole cell recording measures, it was found that bath application of 5-HT induced a postsynaptic inhibitory outward current in some neurons, attenuated glutamate release from the RHT, reduced GABA release and even produced an excitatory current in a small population of rat SCN neurons (Jiang et al., 2000). Both reversible translation and transcriptional inhibitors appear to block the phase shifting effects of 5-HT and its agonists in vitro however it is not yet known which proteins transcriptions, if any are activated by 5-HT at the SCN (Jovanovska and Prosser, 2002).
Comparable to the rat, 5-HT and 8-OH-DPAT both induced 2-3 hr midday phase advances with no effects at other times in mouse brain slices and this effect was blocked by metergoline (Prosser, 2003). In vitro studies of 5-HT7 receptor knockout mice brain slices compared to wildtypes has revealed similar ZT6 advances with bath application of
8-OH-DPAT and that these phase advances are not blocked by 5-HT2C antagonism, but are blocked by 5-HTiA antagonism (Sprouse et al., 2005). This suggests that in mice, 8-
OH-DPAT acts on the 5-HTiA receptors. 5-HT]A knockout mice also fail to shift to 8-
OH-DPAT (Smith et al., 2008). It has also been found that pre-treatment of mouse SCN slices with compounds that increased 5-HT signaling such as 8-OH-DPAT, or fluoxetine 22 blocked or attenuated phase shifting to later 5-HT or 8-OH-DPAT administration
(Prosser et al., 2006). The implication of this study opens the possibility of receptor upregulation due to reduced serotonin signaling making in vitro brain slices supersensitive to serotonergic stimulation. This may explain some of the differences seen between in vivo and in vitro studies.
1.9 Evidence Contrary to the Role of 5-HT
Despite the number of studies implicating 5-HT in non-photic phase resetting, there are also several key findings inconsistent with this hypothesis. One possibility that cannot be excluded is that different non-photic stimuli act on the SCN through at least partially distinct pathways (see Mistlberger et al., 2000). There may be subtle species differences in non-photic signaling as well. Arguing against a direct role of 5-HT in non- photic phase shifting, 5,7-DHT lesions of the SCN fail to abolish midday phase shifts to novel wheel access in responders (Cutrera et al., 1994; Bobrzynska et al., 1996b; Meyer-
Bernstein and Morin, 1998). In fact, a complete lesion of all the 5-HT cells in the MRN also fails to abolish midday phase shifts to novel wheel access in responders (Bobrzynska et al., 1996b). Exposure to a novel wheel in the early part of the active period has also been shown to actually suppress the already high levels of 5-HT release and still result in a phase delay (Dudley et al., 1998). An interpretation of this finding could be that an increase in 5-HT release is not necessary for a non-photic phase shift to occur. Quipazine shifted the SCN of rats in a manner more similar to that of light than non-photic stimuli
(Kohler et al. 1999). In vitro work using isolated rat SCN slices has found similar results 23 indicating 5-HT may have more of a photic-like effect on the rat SCN although this effect seems to vary across strain (Kalkowski and Wollnik, 1999).
Other pharmacological studies have found that mice failed to shift to systemic administration of 8-OH-DPAT across a wide range of time points (Antle et al., 2003).
Some studies reported quipazine fails to shift the hamster circadian clock when applied in the middle or later subjective day (Bobrzynska et al., 1996a; Antle et al., 2003). Despite the findings with the bilateral injections and timed perfusions, unilateral injections of 8-
OH-DPAT directly into the SCN also fail to shift the clock (Mintz et al., 1997; Antle et al., 2003). This suggests that 8-OH-DPAT is not acting directly on the SCN to produce phase shifts as raphe injection of 8-OH-DPAT produces phase shifts (Mintz et al., 1997).
Various serotonin antagonists all failed to attenuate phase shifts to novel wheel access, or shift hamster rhythms when administered alone (Antle et al., 1998). Drug treatments that increased serotonin release in the SCN also failed to shift the clock (Antle et al., 2000).
Even injections of a 5-HT precursor, which has been shown to increase SCN 5-HT levels by over 200%, does not phase shift, or potentiate phase shifts to novel wheel access
(Mistlberger et al., 2000). These findings are at odds with the fact that electrical activation of the raphe produces midday phase advances and brings the question of which transmitter is involved in these shifts (Meyer-Bernstein and Morin, 1999).
1.10 Current Studies
Due to mixed results and inherent species differences in the effects of 5-HT on the circadian system, it is still not clear whether 5-HT acts as the mechanism of non- photic phase shifting at the SCN, or how this occurs in vivo. Since electrical activation of 24 the raphe would activate both the serotonergic and non-serotonergic projections, it is possible that another neurotransmitter is responsible for non-photic like phase shifting.
Therefore, it is useful to examine the yet unidentified projections from the median raphe to the SCN to determine if they play a role in modulating circadian phase. If so, then this may help explain the seemingly disparate findings regarding the effect of serotonin on the
SCN.
Recently, vesicular glutamate transporter 3 (VGLUT3) has been localized in serotonergic and non-serotonergic neurons of the hamster raphe nuclei (Mintz and Scott,
2006). VGLUT3 was present in cell bodies, as well as terminals and astrocytes in most of the DRN and MRN.
1.11 Vesicular Glutamate Transporters
Glutamate neurotransmission requires specialized synaptic vesicles for sequestering and exocytotic release into the synapse known as the VGLUTs. Glutamate synthesis occurs in the cytoplasm and two types of transporters originally thought to be responsible for phosphate movement were found to pack glutamate into vesicles for exocytotic release (Fremeau et al., 2001). VGLUT1 is greatly expressed in the cortex, limbic system, and importantly the SCN. VGLUT2 has been localized as largely complimentary of VGLUT1, predominately in the brainstem and posterior hypothalamus where it is thought to modulate histaminergic transmission (Herzog et al., 2001; Ziegler et al., 2002). VGLUT1 is present in the bipolar cells and VGLUT2 in the retinal ganglion cells colocalized with a subset of melanopsin expressing neurons where they are thought to participate in photoentrainment (Johnson et al., 2007). Another localization study has 25 also shown VGLUT3 to be present in the cholinergic interneurons of the striatum, caudate putamen, accumbens, and both raphe nuclei (Gras et al., 2002). In the rat, a high level of VGLUT3 mRNA is present in the MRN and DRN, there is some colocalization in GABAergic interneurons of the hippocampus, but is absent from the hypothalamus
(Herzog et al., 2004). The use of VGLUTs to unambiguously identify neurons that release glutamate is necessary as glutamate itself is an intermediary step in other important neurochemical processes (Cooper et al., 2003).
It has not yet been determined whether VGLUT3 positive cells in the median raphe project to the SCN. These VGLUT3 cells may serve as a marker of excitatory neurotransmission and could indicate the phenotype of the yet unknown fibers projecting from the MRN to the SCN. Expression of VGLUT3, GABAergic markers and tryptophan hydroxylase staining in the medullary raphe has been examined. Heavy staining for all three were seen in bulbospinal raphe neurons with a large percentage of those being triple labelled and the synapses lacked a consistent pattern with approximately equal numbers of asymmetric and symmetric synapses (Stornetta et al., 2005). This suggested that these neurons might be GABAergic, glutamatergic, or both. Recently, it was reported that the anterior paraventricular nucleus of the thalamus projects to the SCN and co-releases both
GABA and glutamate (Javier and Raul, 2008). Therefore, if MRN VGLUT3 cells project to the SCN, it is possible they may co-transmit glutamate and GABA along with the serotonergic fibers.
Previous results from our labs seem to indicate some of these VGLUT3 positive cells are non-serotonergic and were found to project to certain layers of the hippocampus
(Jackson and Antle, 2008). The purpose of this study is to determine whether VGLUT3 26 cells from the MRN project to the SCN through retrograde tract tracing. Next, we wanted to examine whether these non-serotonergic projections were important for non-photic phase shifting through electrical activation of the raphe in animals with selective serotonergic lesions of the SCN.
Chapter Two: Retrograde Tract Tracing
2.1 Introduction
The MRN and DRN are the primary sources of 5-HT in the mammalian forebrain
(Jacobs et al., 2002). The MRN sends a major projection to the SCN, about half of which is serotonergic (Meyer-Bernstein and Morin, 1996). Electrical stimulation of the MRN during the midday produces small phase advances, with small phase delays occurring during the early night similar to the non-photic PRC (Meyer-Bernstein and Morin, 1999).
Electrical stimulation of the MRN should equally activate both serotonergic and non- serotonergic projections to the SCN. It has not yet been determined if activation of the serotonergic, or non-serotonergic projections is necessary to produce these non-photic phase shifts. The non-serotonergic projections to the SCN have not been well characterized. Recently, VGLUT3 has been colocalized in serotonergic and non- serotonergic cells of the hamster raphe (Mintz and Scott, 2006). The goal of this experiment is to determine if raphe cells containing VGLUT3 project to the SCN in order to better characterize some of the non-serotonergic projections to the SCN. It was 27 hypothesized that cells that contain VGLUT3 but do not contain serotonin project to the
SCN.
2.2 Animals and Apparatus
Male Syrian hamsters were obtained from Charles River laboratories (Kingston,
NY). Animals were initially group housed 2-3 per cage. They were first acclimatized to laboratory conditions in a 14:10 LD cycle. Following surgical procedures, animals were single housed. All surgical procedures took place in a Kopf stereotaxic frame with the incisor bar set to -2.0 mm below the interaural line. Care was taken to avoid pain and discomfort for the animals used in the study. The Life and Environmental Sciences
University of Calgary Animal Care Committee approved all procedures. Ethical protocols were in accordance with the guidelines of the Canadian Council of Animal Care.
2.3 Experiment 1
2.3.1 Cholera Toxin P Subunit Ionotophoresis
Hamsters (n=28) were pre-treated with a subcutaneous injection of the analgesic
Torbugesic 2 mg/kg (Butorphanol Tartrate; Wyeth Canada, Que). Next they were anesthetized with sodium pentobarbital (85-100 mg/kg; Ceva Sante Animale, France) i.p. and placed in the stereotaxic frame. A glass electrode with a 20-30 um tip on was directed at the SCN on a 10° angle using the following coordinates from bregma: anterior/posterior (AP): +0.6, medial/lateral (ML) +1.25, and dorsal/ventral (DV) -7.65 28 mm from the dura. A 10% iridium platinum wire 0.2 mm in diameter was immersed in the cholera toxin P subunit (CTb) (1% in distilled water; List Biological Laboratories) and attached to the lead while the ground was attached to a skin flap on the skull. The glass electrode was lowered using a continuous negative retaining current of -1.0 tiA from a Ration Scientific BAB-150 iontophoresis pump. Once in place, a direct positive current of 1.5 uA was applied for 15 minutes using 5-second pulses to eject the tracer.
The electrode was left in place for 5 minutes after iontophoresis to minimize flow-back during withdrawal. A negative retaining current of-1.0 |xA was used during withdrawal of the electrode to avoid leakage along the track. Animals were sutured and allowed to recover for 7-10 days. Due to the relatively fast transport time of CTb, (4.25 mm/h or 102 mm/day) this would have given the tracer ample time to reach the MRN (Wu et al.,
1999).
At the conclusion of the study, animals were then deeply anesthetized with sodium pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, ON, Canada) and perfused transcardially using 100 ml cold phosphate buffered saline pH 7.4 followed by
100 ml cold 4% paraformaldehyde. Brains were post fixed over night and then cryoprotected using 20% sucrose in PBS for one more day. Tissue was sliced at 35 urn on a Leica Cryostat at -18°C and alternate sections collected into wells of PBS for immunocytochemistry (ICC) in order to visualize the injection site and tracer spread.
2.3.2 SCN Cholera Toxin Immunocytochemistry
Alternate sections of the SCN were collected into wells for ICC procedures. To begin, tissue was incubated in 0.5% H202 in PBSx (0.3% Triton X-100 in PBS) for 15 29 minutes on a shaker tray. The tissue was rinsed in PBSx and then incubated in a blocking solution of 10% normal horse serum (Vector Laboratories Inc., Burlington, ON, Canada) in PBSx for 90 minutes. The primary antibody used was a goat anti-cholera toxin
(1:3000; List Biological Laboratories, CA, USA) in 3% normal horse serum in PBSx.
Primary incubation took place for 48 hours at 4°C on a shaker tray. Sections were rinsed and incubated in the secondary biotinylated horse anti-goat (1:200; Vector Laboratories
Inc) for 60 minutes. Tissue was rinsed again and incubated for 60 minutes in an avidin- biotin complex (ABC; 1:100; Vector ABC Elite Kit; Vector Laboratories Inc) in PBSx before being rinsed again. Visualization of the ABC complex was accomplished using
0.05% diaminobenzidine (DAB; 0.5mg/ml) as a chromagen in 0.1M Tris-buffer with 80 ul of 30% H2O2. 0.02% nickel chloride was used for intensification of the reaction product. Tissues sections were mounted onto gelatin coated slides, dehydrated in a successive series of alcohol rinses, cleared in xylene and then cover slipped with
Permount (Fisher Scientific, Pittsburgh, PA, USA).
Some alternate series were collected and processed for fluorescent immunocytochemistry (see below). One of the series was used to visualize the site of iontophoresis using Cyanine (Cy)3 conjugated donkey anti-goat (Jackson
ImmunoResearch, Laboratories, Inc., West Grove, PA, USA).
2.3.3 5-HT-VGLUT3-Cholera Toxin Immunocytochemistry
Alternate 35 um sections were obtained using a cryostat through the entire rostral caudal extent of the MRN of those animals receiving successful iontophoresis if CTb into the SCN. The sections were rinsed and then incubated in 4% normal donkey serum in 30
PBSx for one hour at room temperature. Primary antibodies used were goat anti-cholera toxin (1:3000; List Biological Laboratories), guinea pig anti-VGLUT3 (1:3000;
Chemicon International, CA, USA) and rabbit anti-5HT (1:2500; Immunostar, Hudson,
WI, USA) in 2% normal donkey serum in PBSx. Primary incubation took place on a shaker tray at 4°C for 48 hours. Tissue was then washed 6 times and left in a shaker tray overnight in PBSx at 4°C. On the final day, tissue was rinsed 6 times again and then protected from light from that point on. Slices were incubated in the fluorescent secondary antibodies for one hour (1:200 Cy2 conjugated donkey anti-rabbit, Cy3 conjugated donkey anti goat, and Cy5 conjugated donkey anti-guinea pig; Jackson
Immunoresearch, PA, USA) Finally, the tissue was mounted, dehydrated, cleared in xylene and then cover slipped with Krystalon (VWR International, Edmonton, AB,
Canada).
2.3.4 Analysis
Success of ionotophoresis of CTb was determined by examining the CTb immunoreactivity at the SCN. Label had to be largely within the SCN, with minimal spread beyond the SCN boundaries. One series of sections throughout the entire rostral caudal extent of the MRN was photographed using an Olympus BX51 microscope equipped with a cooled CCD camera (QICAM 1394; Qlmaging, Surrey, BC, Canada).
For the fluorescent labelled sections, high intensity light was passed through filters to image each of the three labels. Cells were counted in Adobe Photoshop (Adobe Systems,
San Jose, CA, USA). First it was determined which cells contained CTb labelling by examining that specific color channel. Next it was determined whether these cells were 31 immunoreactive for 5-HT and/or VGLUT3 by examining each of the other color channels. To be considered a triple label immunoreactive cell, labelling clearly had to match the shape of the same cell in all three-color channels. Mean cell counts are reported +/- standard error.
2.4 Results
A total of n=5 animals were determined to have received successful iontophoresis of CTb into the SCN. There was one case (Figure 2.1 A) in which, heavy unilateral labelling was found in the dorsal raphe in an area not immediately adjacent to the fourth ventricle with only a few fibers apparent in the ependymal layer, a pattern not consistent with ventricular leakage (Chen et al., 1999). In all other cases, (Figure 2.1 B-E) very few cells were found in the DRN indicating ventricular leakage likely did not occur. In another case, (Figure 2.1 E) there was some unilateral infusion of CTb into the optic chiasm in the rostral part of the SCN that did not occur in any other case. In two instances, (Figure 2.1 A and E) there were several labelled cells seen along the electrode track indicating some leakage along the track. Since track labelling was sparse and there were no consistent differences between these two cases and the others, it was assumed not to affect the results.
A representative triple labelled MRN section is displayed (Figure 2.2 A). Cells containing the retrograde tracer were also apparent in the paramedian raphe of some animals. The paramedian raphe is a loose conglomeration of 5-HT containing cells surrounding the dense MRN throughout the rostral caudal extent. Four types of cells in the raphe were found to project to the raphe. The first cell type apparent was single- 32 labelled CTb cells (Figure 2.3 A). As expected there were cells that double labelled for
CTb and 5-HT (Figure 2.3 B). Cells also double labelled for CTb and VGLUT3 (Figure
2.3 C). The final type of cell found was triple labelled for CTb, 5-HT and VGLUT3
(Figure 2.3 D).
The first case (Figure 2.1 A, Figure 2.4 A) had an electrode tip and the most intense cholera toxin staining in the core region of the SCN where there are calbindin containing neurons. There was relatively very little spread lateral and dorsal of the SCN.
Some cells labelled in the SCN contralateral to the iontophoresis site through passive transport. Iontophoresis of the retrograde tract tracer in this region resulted in intense unilateral staining of the DRN (Figure 2.4 B) as is discussed below. This case also resulted in labelling of the MRN with a majority of the projection consisting of approximately 38% single labelled CTb neurons. Only about 32% of the projection contained serotonin and around 30% contained VGLUT3 but not serotonin.
Three cases appeared to have an electrode tip and the most intense CTb staining in the shell of the SCN. In one of those cases, (Figure 2.1 B) iontophoresis of the CTb was largely confined to the SCN with some spread dorsal and caudally. The area with the densest fluorescent staining was located immediately ventral and slightly lateral to the third ventricle in the shell of the SCN. This case (Figure 2.1 B) was found to consist of a higher proportion of cells containing VGLUT3 but not serotonin at around 43% of the projection. Only about 26% of the projection single labelled for serotonin and 30% was single labelled for CTb. This case was similar to another animal (Figure 2.1 D) in which the most intense labelling was observed ventral to the third ventricle still in the dorsomedial shell of the SCN. There was slight spread anterior, some spread lateral, 33 dorsal and caudal from the SCN. In this case approximately 28% of the MRN projection to the SCN labelled for cholera toxin, around 56% single labelled for serotonin and only
16% labelled for VGLUT3 but not serotonin. Another case (Figure 2.1 C) also appeared to label in the dorsal shell region, slightly lateral to the base of the third ventricle. There was some spread into the medial preoptic nucleus, rostral to the SCN and tracer spread into the retrochiasmatic area caudally. Within the SCN, there was some spread of the tracer dorsally and laterally. Cells projecting to the SCN in this case appeared to be equivalent across all types with the highest proportion (-33%) coming from cells that only expressed VGLUT3. The remaining three cell types each comprised around 20% of the projection.
In the final instance, the most intense CTb staining was found at the ventromedial aspect of the SCN in an area corresponding to the area of densest serotonergic innervation. There was some spread of the tracer into the optic chiasm and hypothalamic area rostral to the SCN. In this instance, (Figure 2.1 E) approximately 32% of the MRN projection was comprised of triple label neurons for 5-HT, VGLUT3 and CTb. About
25% labelled for 5-HT with an equal proportion single labelling for VGLUT3. The remaining 18% was found to only contain the tracer.
2.4.1 Median Raphe and Paramedian Raphe Labelling
An average of 46.6 ± 2.8 cells were labelled with cholera toxin in one series of raphe sections for those animals receiving successful iontophoresis of CTb into the SCN
(Figure 2.2 B). Cells containing the retrograde tracer were also apparent in the paramedian raphe of some animals according to the hamster brain atlas (Morin and 34
Wood, 2001). Since very few cells on average actually labelled in the paramedian raphe, those cells were grouped together with median raphe labelled cells. Of the cells labelled in the median and paramedian raphe then, an average of 13 ± 1.9 were single labelled for cholera toxin, 9.8 ± 1.2 double labelled for CTb and 5-HT, 14 ± 2.9 double labelled for
CTb and VGLUT3 and 9.8 ± 1.2 triple labelled for CTb, 5-HT and VGLUT3. Combined then, approximately 42.1% of the projection from the median raphe to the SCN was serotonergic which is consistent with previous results that estimated approximately 50% of the projection was serotonergic (Meyer-Bernstein and Morin, 1996). Approximately
30% of the projection stained for VGLUT3 but not 5-HT indicating a substantial contribution of neurons containing a marker for glutamate. Finally, 27.9% of the projection single labelled solely for the tracer indicating approximately a third of the projection from the MRN to the SCN was a yet an unknown cell type. In total then, about
72.1%) of the projection from the MRN to the SCN could be accounted for by 5-HT and
VGLUT3 alone.
2.4.2 Dorsal Raphe Labelling
As mentioned above, dorsal raphe neurons labelled inconsistently and did not appear to be the result of inadvertent leakage of the tracer into the ventricle (Chen et al.,
1999). Early evidence from lesions and electron microscopy indicated that the rat raphe nuclei innervated the ependymal wall of the ventricular system as well as brain regions
(Aghajanian and Gallager, 1975). Thus, false positive retrograde labelling in the raphe would be apparent if there was tracer leakage into the ventricles, or ependymal wall.
There was one case (Figure 2.4 A also displayed in Figure 2.1 A) in which 107 cells 35 labelled for CTb in the DRN, which was more than twice that observed in the MRN. The iontophoresis electrode tip appeared to be localized in the core of the SCN where tracer staining was the most intense (Figure 2.1 A). Within the DRN intense retrogradely labelled cell bodies were situated in the ventrolateral part of the DRN (Figure 2.4 B). The remaining cases (Figure 2.1 B-E) labelled with 20 cells or less in the DRN, far less than the number of cells labelling in the MRN. Of those animals that received successful iontophoresis into the SCN then, an average of 29.4 ± 19.6 cells were retrogradely labelled in the DRN (Figure 2.5). Of those DRN tracer-labelled neurons, 7.6 ± 3.8 cells labelled for CTb only, and accounted for 25.9% of the projection, 10.8 ± 8.6 cells double labelled for CTb and 5-HT and accounted for 36.7 % of the projection, 4.4 ± 2.5 double labelled for CTb and VGLUT3 accounting for 15% of the projection and finally, 6.6 ±
5.0 cells triple labelled for CTb, 5-HT and VGLUT3 accounting for about 22.4% of the projection. In sum then, a little over 59% of the projection was serotonergic and only about 15% was judged to contain VGLUT3 but not serotonin. Contrary to previous findings, all of our successful tracings from the SCN (n=5) as well as some partial hits in the SCN that were examined showed some labelling in the DRN (n=6) for a total of 11 animals.
2.5 Discussion
2.5.1 Retrograde Tract Tracing
Overall, these results are consistent and complimentary to that of previous studies on the MRN projection to the SCN. We replicated and extended findings that only about 36 half of the MRN projection to the SCN contains serotonin. Unlike previous findings, we found the projection from the median raphe to the SCN was an average of 46.6 ± 2.8 cells from counting alternate 35 urn sections through the MRN. Therefore our estimate of the entire MRN projection would be, on average, 94 cells, which is approximately twice that which was previously estimated when counting every fourth slice (Meyer-Bernstein and
Morin, 1996). We also extended observations on the types of cells projecting to the MRN in finding that cells containing VGLUT3 but not 5-HT also project to the SCN. VGLUT3 was also present in serotonin containing raphe cells that project to the SCN. In total then cells containing serotonin and/or VGLUT3 comprise 72.1% of the total projection from the median raphe to the SCN.
Although it has been reported that VGLUT3 is present in almost all of the raphe serotonergic cells, only several forebrain areas have been found to contain double label fibers for VGLUT3 and 5-HT (Shutoh et al., 2008). This suggests that there are at least two types of fibers the project from serotonergic raphe cells, most containing only serotonin or that not all cells contain VGLUT3. In this study we found approximately
48.9% of the projection from the median raphe to the SCN did not appear to contain
VGLUT3. On average only about 23 cells were judged not to contain VGLUT3. This is a relatively small proportion of the estimated 1100 cells in the MRN. Previous findings indicate nearly all of the serotonergic neurons in the hamster raphe contained some level of VGLUT3, although in some cases levels were very low (Mintz & Scott, 2006). The alternative explanation is that our procedures were not as sensitive as previous findings that used confocal microscopy, or that our criteria for determining whether VGLUT3 was present in a cell were too stringent (for a discussion see Jackson et al., 2008). In our study 37 a cell was not deemed to be VGLUT3-immunoreactive unless label was clearly seen outlining the soma.
We also report inconsistent but highly unusual DRN staining following retrograde tracing from the SCN. In general there is a consensus that the sole source of 5-HT in the
SCN comes from the median raphe however, one older tracing study found equal numbers of sparsely distributed neurons in the DRN and MRN of the golden hamster following retrograde tracing from the SCN (Pickard, 1982). Another retrograde tracing study found several fibers seen at the dorsal tip of the SCN following anterograde tracing from the DRN (Leander et al., 1998) In the rat, some retrograde labelling has revealed a partially serotonergic projection from the DRN to the SCN (Kawano et al., 1996). In a species of diurnal shrew it has been reported that the ventrolateral DRN cells contribute directly to SCN serotonergic innervation (Su and Liu, 2001). Interestingly enough, the ventrolateral portion of the DRN was where retrogradely labelled neurons were found in one of our cases (Figure 2.5 B). It has been argued that older studies reporting a DRN to
SCN projection were complicated by spread of the tracer into hypothalamic areas rostral to the SCN (Morin and Meyer-Bernstein, 1999). The former studies where no DRN labelling was found have the advantages of being both retrograde and anterograde studies, even including serotonergic lesions in order to assess location of serotonin fiber loss. Our results have the advantage of further elucidating the heterogeneity within the
SCN based on subtly different ionotophoresis sites. These different cell subpopulations within the SCN gave rise to varying retrogradely labelled subpopulations in both the
MRN and DRN. Of particular interest was the unilateral DRN labelling found following unilateral retrograde tract tracer application in the core region of the SCN that did not 38 seem to be replicated in any of our other cases where core SCN iontophoresis was not apparent.
2.5.2 The Role of VGLUT3
It has yet to be determined whether VGLUT3 is a good marker of cells that engage in synaptic glutamate neurotransmission. It was initially thought that VGLUT1 and 2 could account for all synaptic glutamatergic neurotransmission. While VGLUT1 and 2 are situated almost solely on terminals, VGLUT3 is expressed on cell bodies, dendrites, presynaptic terminals and astrocytes making its role all the more unclear (Seal and Edwards, 2006a). VGLUT1 and VGLUT2 are present in the rat dorsal raphe where
VGLUT2 is more numerous and contacts dendritic shafts whereas VGLUT1 mainly contacts more distal dendritic spines (Commons et al., 2005). This indicates a complex glutamatergic modulation of raphe firing, but it has not yet been conclusively demonstrated that the raphe in turn sends glutamatergic projections elsewhere. An extensive study on VGLUT3 localization in the rat brain revealed evidence that VGLUT3 is involved in a synaptic release of glutamate due to its similarities in glutamate transport with VGLUT1 and 2, as well as a role in retrograde synaptic signaling due to its localization on dendrites and cell bodies (Fremeau et al., 2002).
Since VGLUT3 was found inside the membrane concentrated along the cell body it has been suggested that VGLUT3 could be storing glutamate for release into the extracellular space (Mintz and Scott, 2006; Takamori, 2006). This could allow for the raphe to have the ability to excite itself. Glutamate levels in the SCN oscillate in a circadian manner that persists with the application of tetrodotoxin (Rea et al., 1993). The 39 same sort of mechanism that does not rely on action potentials could be operating in the raphe using VGLUT3 (for a discussion see Mintz and Scott, 2006). It could also be the case that some cells containing VGLUT3 release glutamate from terminals, and others are used for autocrine or paracrine excitation of raphe cells. If VGLUT3 can be used as a reliable marker of synaptic glutamatergic neurotransmission then another source of glutamate is present in the SCN outside of the already well-established source from retinal terminals (Morin and Allen, 2006). It has been established that the SCN contains all of the examined ionotropic glutamate receptors, so the idea that glutamate has more than one role in the SCN does not seem unreasonable (Stamp et al., 1997). How one neurotransmitter could produce both the photic and non-photic PRC would be another question though. It is also of note that about a third of the projections single labelled for cholera toxin. This indicates a substantial proportion still remains unknown. While many attempts have been made to localize other neuromodulators, or neurotransmitters in the raphe, none to our knowledge have attempted to assess their relative contributions to
SCN innervation. It may be the case cells containing VGLUT3 contribute to non-photic phase shifting in the SCN, or these yet unknown single label CTb cells are involved.
It has been suggested that some of the non-serotonergic raphe cells in the rat may be dopaminergic and project to the hippocampus (Reymann et al., 1983; Trulson et al.,
1985). The neuropeptide galanin was found to colocalize in a dorsal subset of rat median raphe serotonergic neurons (Melander et al., 1986). Vasopressin terminals and fibers were also found throughout the dorsal and median raphe (DeVries et al., 1985). The endogenous acidic dipeptide N-acetyl-aspartyl-glutamate (NAAG) has been colocalized in a subset of median raphe serotonergic neurons as well (Forloni et al., 1987). NAAG is 40 a precursor to glutamate has been proposed as a neurotransmitter on its own at certain receptor subtypes.
More often, it is becoming apparent that cells engage in co-release of neurotransmitters. For example, cells in the dentate gyrus as well as auditory brainstem have been shown to co-release both GABA and glutamate (Seal and Edwards, 2006b).
Triple label VGLUT3, tryptophan hydroxylase, GABAergic neurons have been localized that project to the spinal cord (Stornetta et al., 2005). Recently, it was reported that the anterior paraventricular nucleus of the thalamus projects to the SCN and co-releases both
GABA and glutamate (Javier and Raul, 2008). It has also been found that in microculture, serotonergic raphe neurons induced glutamatergic potentials (Johnson,
1994). This could indicate that the serotonergic VGLUT3 raphe neurons we found co- release either GABA or glutamate. Using double label immunofluorescence, it was determined that cells containing glutamic acid decarboxylase, a marker for GABA were only rarely colocalized with serotonergic raphe cells and that most of the GABA in the raphe probably originates from interneurons, or cells outside of the raphe (Stamp and
Semba, 1995). This provides evidence against GABA being a substance that is co- released from the raphe. The nature of the non-serotonergic cells in the raphe and whether they engage in some kind of co-release can also be questioned.
Cells and fibers in the hamster SCN and surrounding regions were found to colocalize for both tyrosine hydroxylase (TH) and L-amino acid decarboylase, a rate- limiting enzyme in dopamine synthesis indicating the neurons (Novak and Nunez, 1998).
The origin of most of these putative dopaminergic fibers in the SCN is unknown. Our own (unpublished) observations indicate that the hamster dorsal raphe shows a high 41 degree of TH immunoreactivity mostly in fibers but in some cells as well. These TH cells do not appear to colocalize with the serotonergic cells; however it is not yet known whether they colocalize with VGLUT3 positive cells although it can be assumed they do.
Cultured dopaminergic neurons from the rat appeared to make glutamatergic synapses indicating some form of co-transmission (Sulzer et al., 1998). This may indicate that some of the non-serotonergic VGLUT3 containing neurons in the DRN engage in dopaminergic and glutamatergic co-release. TH does not appear to be present in the
MRN.
There are many possible candidates for these non-serotonergic projections important for non-photic shifting, and it may be the case that cells in the raphe use some form of co-transmission. A recent study found that p-ERK was suppressed in the SCN shell of animals that shifted to sleep deprivation (Antle et al., 2008). Furthermore, some of the animals that shifted in Antle et al., (2008) also exhibited an increase in p-ERK in a group of neurons in the SCN core in direct response to the non-photic manipulation. This could indicate that the neurotransmitter involved in raphe modulated non-photic phase shifts should stimulate the phosphorylation of these ERKs in the core SCN. Given that we have found four types of cells in the MRN project to the SCN, and each of these cells may co-transmit several different types of neurotransmitters, it is highly likely that the raphe projection would be able to differentially modulate ERK phosphorylation in the
SCN. Since we have evidence for a direct DRN to core SCN projection, it is even possible that the DRN may modulate an increase in p-ERK while the MRN projection may mediate the p-ERK suppression in the shell. 42
There is much evidence to support a role for the raphe in non-photic phase shifting. The role for serotonin is in doubt. 5,7-DHT lesions of MRN serotonin cells fail to eliminate shifts to novel wheel access (Bobrzynska et al., 1996b). It has been suggested that in the mouse, 5-HT and NPY from the IGL may both be necessary in order to induce non-photic phase shifts (Marchant et al., 1997). It also may be the case that serotonin release in an area outside of the SCN is necessary for the regulation of circadian rhythmicity (Glass et al., 2000; reviewed in Morin and Allen, 2006). Since the raphe projects widely to virtually all areas of the forebrain it is important to know how it utilizes various neurotransmitters. One of the first steps will be clarify the role of
VGLUT3 in order to determine if it can be used a reliable marker of synaptic glutamate neurotransmission. Using the hamster model of non-photic phase shifts, electrical stimulation of the raphe, lesions and pharmacological approaches give us a powerful method for determining the phenotype of raphe cells in order to fully clarify how it communicates with the rest of the brain and provides input to the circadian system.
2.5.3 Methodological Considerations
The very nature of retrograde tract tracing lends itself to several methodological criticisms. These criticisms would involve actual distribution of retrograde tracer into the area of interest. One might argue that unless there was diffusion of the tracer throughout the entire structure of interest, then it cannot be certain that all afferent inputs into that area can be accounted for. The converse is true, if too much retrograde tracer is applied to the structure and there is leakage into the surrounding structures, it cannot be said for certain whether certain afferent inputs are to the structure of interest itself. The whole 43 debate as to whether the DRN projects to the SCN could be an example of a highly selective input that has escaped notice in other studies. These other studies have the advantage of using both retrograde and anterograde tract tracing techniques. In our study, the use of a small electrode iontophoresis electrode (20-30 um) was serendipitously advantageous. We were able to have very specific iontophoresis sites within the SCN that may not be apparent in the whole structure approach used in other studies. The SCN is composed of a very heterogeneous structure where different subsets of neurons work in concert to perform daily timekeeping tasks (Antle and Silver, 2005). Due to the nature of cellular organization in the SCN, this more specific subregion tracing approach may be necessary to tease apart subtle differences in inputs. Using fluorescent methods it was possible to determine where the dense core of tracer infusion was located within the
SCN. This core was where the iontophoresis electrode tip was situated and was usually surrounded by a less dense shell of the tracer that probably resulted from passive diffusion. It seems likely that terminals within the dense core region of the tracer injection site should take up sufficient retrograde tracer to show retrograde tracing, it is less clear to what extent this occurs in the shell region of the tracer injection site. The usage and reporting of more specific retrograde tract tracing iontophoresis sites may be advantageous in determining more specific afferent inputs of interest.
Chapter Three: MRN Stimulation 44
3.1 Introduction
The midbrain MRN sends a substantial serotonergic projection to the SCN
(Meyer-Bernstein and Morin, 1996). Electrical stimulation of the MRN phase shifts the
SCN in a non-photic phase dependent manner (Meyer-Bernstein and Morin, 1999). Large increases in 5-HT release at the SCN has been reported during exposure to non-photic manipulations implicating 5-HT as a key factor in inducing non-photic phase shifts
(Dudley et al., 1998; Grossman et al., 2004; Mendoza et al., 2008).
There is some conflicting evidence as to whether 5-HT is involved in these phase shifts though. Lesions of the serotonergic input into the SCN fail to eliminate shifts to midday novel wheel access (Meyer-Bernstein and Morin, 1998). Direct, unilateral application of 8-OH-DPAT directly into the SCN also fails to shift the clock (Mintz et al.,
1997; Antle et al., 2003). Administration of various 5-HT antagonists also fails to attenuate shifts to novel wheel access (Antle et al., 1998). Selective pharmacological activation of MRN 5-HT fibers that results in large increases of 5-HT release at the SCN also fails to shift the clock (Antle et al., 2000).
Since the MRN projection to the SCN is only about 50% serotonergic, it may be the case that the non-serotonergic part of the projection is activated by MRN stimulation and releases a yet unknown neurotransmitter that results in, or modulates non-photic phase shifts. The goal of this study is to determine if intact serotonin input is necessary for midday phase advances to electrical activation of the MRN. Intact animals receiving electrical stimulation of the raphe at midday were hypothesized to show a phase advance.
Animals receiving a lesion to the serotonin input into the SCN were also expected to 45 show a significant phase advance. It was hypothesized that the serotonin input into the
SCN was not necessary for midday phase advances to electrical stimulation of the raphe.
3.2 Experiment 2
Initial pilot work on this study was accomplished using a within-subjects design.
Animals (n=12) received MRN electrode implants as described below, but also received simultaneous implantation of a 22-gauge stainless steel cannula to the SCN (HRS
Scientific, Montreal, Que, Canada). SCN cannulas were directed at following coordinates from bregma on a 10° angle laterally: AP +0.6, ML -1.3, DV -6.7 mm from dura. The injector tip would extend 1 mm beyond the end of the cannula to reach the SCN. The incisor bar was set to -2.0 mm below the interaural line.
These animals were subjected to electrical stimulation procedures as described below, but also received control stimulation procedures where they were connected to the stimulation apparatus, but no current was passed. Manipulations were performed in a counterbalanced fashion with half of the group receiving a control stimulation procedure first. After receiving a control and stimulation manipulation, animals were briefly removed from DD to conduct sham control and selective 5-HT lesions in the SCN. All animals received an i.p injection of desipramine hydrochloride (25 mg/kg dissolved in physiological saline; Sigma-Aldrich, Canada, Oakville, ON) to protect the catecholaminergic terminals (Gerson et al., 1974). 30 minutes before receiving a central injection either vehicle (0.5% ascorbic acid in physiological saline) or 5,7-DHT (~25 ug free base in 0.5% asorbic acid in sterile saline; Sigma-Aldrich, Canada, Oakville, ON) through the cannula. Animals were monitored in the light following injection and then 46 returned to DD. Free running rhythms in DD were once again established and then electrical stimulation of the raphe repeated.
3.3 Experiment 2B
Due to experimental design issues in experiment 2, an additional between subjects design was accomplished (discussed below). 30 minutes prior to receiving an anesthetic for surgery, lesion animals (n=6) were administered a systemic injection of desipramine hydrochloride (25 mg/kg dissolved in physiological saline; Sigma-Aldrich,
Oakville, ON, Canada). Animals (n=12) were anesthetized using sodium pentobarbital
(-70 mg/kg; Ceva Sante Animale, France). A 26-gauge 1 ul Hamilton syringe was directed at the SCN on a 10° lateral angle using the following coordinates from bregma:
AP +0.3, ML +1.25, DV -7.65 mm from dura. Half of the animals (n=6) received a 1 ul
SCN injection of vehicle. The other half (n=6) received a 1 ul injection of 5,7-DHT to lesion the serotonergic input into the SCN (-25 ug free base in 0.5% asorbic acid in sterile saline; Sigma-Aldrich, Canada, Oakville, ON). Animals were then stereotaxically implanted with stainless steel insulated twisted bipolar electrodes (MS303/3, Plastics
One, Inc, Roanoke, VA). The electrode tips were separated slightly and approximately
0.25 mm were exposed. With the incisor bar at -2.0 mm below the interaural line, electrodes were implanted at a 20° lateral angle to avoid obstruction of the cerebral aqueduct. The following coordinates from bregma were used: AP -4.4, ML +2.3, and DV
-6.4 mm from dura. The electrodes were secured using screws drilled into the skull as anchors for a dental acrylic headcap (Dentsply,York, PA). Animals received a 47 subcutaneous injection of Torbugesic (2 mg/kg; Butorphanol Tartrate; Wyeth Canada,
Que) as an analgesic near the end of the surgical procedures.
3.4 Experimental Procedures
Following surgery and 2-4 days of recovery time, animals were released into DD in 45x24x19cm clear polypropylene cages equipped with running wheels 14 cm in diameter attached to the cage lid at one end. The running wheel was connected to a magnetic switch that continuously collected revolution counts that were summed in 10- minute bins on a Dell PC desktop computer using ClockLab data collection software
(Actimetrics, Evanston, IL, USA). Food and water was available ad libitum. Once a stable free running rhythm was established (7-10 days), activity onsets were calculated using the ClockLab data analysis software. A regression line was fit to these onsets and used to predict CT4 for the experimental day.
Electrical stimulation of the median raphe took place from CT4-6 in the activity rhythm (starting 8 hours before predicted activity onset). These circadian times were chosen based on pilot work done in our lab. The experimenter entered the room with the aid of infrared viewing goggles, removed the cage lid, placing the animal in the cage base in the stimulation apparatus. The apparatus was set up on a wooden shelf placed on the floor of the recording room. Animals were connected to a Grass SD88 stimulator coupled to a photic stimulus isolation unit (PSIU6) immediately prior to stimulation (Grass
Instrument Company, Quincy, MA). A constant current of 300-400 uA in 2 ms biphasic pulses (2 ms anodal, 2 ms off, 2 ms cathodal) at 20 Hz was applied for two hours in the home cage. These stimulation parameters were chosen to be consistent with previous 48 studies (Meyer-Bernstein and Morin, 1999) and also based on the results of our own pilot data. The animals were allowed to freely move around the cage bottom while stimulation was taking place.
Following electrical stimulation of the raphe, animals were left undisturbed in DD for 3 days following the manipulation to account for transients and an additional 7 days to calculate a post-stimulation regression line.
3.4.1 Perfusion and Immunocytochemistry
At the conclusion of the study, animals were deeply anesthetized with sodium pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, ON, Canada) and perfused transcardially using 50 ml cold PBS pH 7.4 followed by 50 ml cold 4% paraformaldehyde. Brains were post fixed over night and then cryoprotected using 20% sucrose in PBS for one more day. Tissue was sliced at 35 urn on a Leica Cryostat at -
18°C and alternate sections collected into wells of PBS for ICC in order to visualize the extent of the lesion and electrode tip placements.
In brief, as most of this procedure is described above, alternate sections of the
SCN and MRN for the experimental animals were rinsed, and then blocked using 10% normal horse serum for 90 min (Vector Laboratories Inc., Burlington, ON, Canada). Next tissue was placed in a primary antibody incubation of goat anti-5-HT (1:2500;
Immunostar, Hudson, WI, USA) in 3% normal horse serum in PBSx for 48 hrs at 4°C on a shaker tray. Sections were rinsed and incubated in biotinylated horse anti-goat (1:200;
Vector Laboratories Inc) for 60 minutes. Sections were rinsed again before being blocked in 0.3% H2O2 in PBSx for 30 minutes and then rinsed again. Finally, sections were 49 incubated in the ABC complex for 60 min before being rinsed again (1:100; Vector ABC
Elite Kit; Vector Laboratories Inc). Tissue was reacted using 0.05% DAB (0.5 mg/ml) as a chromagen in 0.1M Tris-buffer with 80 ul of 30% H2O2. 0.02% nickel chloride was used for intensification of the reaction product. Brain sections were mounted onto gelatin coated slides, dehydrated in a successive series of alcohol rinses, cleared in xylene and then cover slipped with Permount (Fisher Scientific, Pittsburgh PA).
3.4.2 Analysis
Phase shifts were calculated by using the ClockLab data analysis software
(Actimetrics, Evanston, IL, USA) to calculate activity onsets for the 7 days immediately prior to the manipulation day. The software calculated the activity onset as the first hour of activity bins exceeding the 50th percentile of average activity following 5 hours of activity below the 50th percentile. A regression line was fitted to these activity onsets and used to predict CT12 for the manipulation day. Following the manipulation day, 3 additional days were left to account for the possibility of transients in the activity rhythms. Activity onsets for the next 7 days were calculated as described above and a regression line was fitted to these onsets. Phase shifts were calculated as the difference between baseline and post-manipulation regression lines on the day of the stimulation.
Phase shifts are reported in hours ± standard error. Counter balanced pre-lesion control and stimulation conditions in experiment 2 were analyzed for an order effect using a 2- way-ANOVA with condition order and manipulation type as factors. Control and stimulation phase shifts were compared using a dependent group t-test for animals that received both control and stimulation manipulations. In experiment 2B, the vehicle and 50 lesion groups were compared using an independent group t-test. All t-tests were two tailed with the a level set to 0.05.
3.5 Results
3.5.1 Behavior during Stimulation
Overall, the response to electrical stimulation of the median raphe was quite varied. The most commonly observed behavior was leftward circling. With heads turned to the left, the hamsters would remain in place, circling constantly toward the left such that they appeared to be chasing their own tails. This circling behavior would often last for around an hour slowing down until eventually the animal regained normal movement toward the latter half of the stimulation. This circling behavior seemed to be more mediated by forelimb movements than hind limbs. Rightward circling was observed much less often. The next most commonly observed behavior was an increase in overall arousal. Some animals would show increased respiration, and bouts of increased motor activity, often running around the outside of the cage and jumping up the sidewalls.
Again, this would often last for at most an hour before the animal appeared to be alert, but no longer showing excessive motor activity. Finally, there were several animals that appeared to show no major behavioral response to the stimulations. In particular, those animals in experiment 2 that received a lesion and subsequent stimulation were more likely to be unresponsive to stimulation. Following most stimulations, animals were fairly unresponsive to physical handling and would often go to sleep right away. Several of the lesioned animals appeared to suffer from poor gait and lack of responsiveness even one 51 day following the stimulation and there was substantial mortality in the lesion group (see below).
3.5.2 Histology
For both experiments, only electrode tips localized in the MRN were used for analysis in the phase shifts (Figure 3.1). This included electrode tips judged to be localized in the paremedian raphe as well as it was assumed current spread would activate raphe cells consistent with behavioral effects observed during the course of the experiments and previous studies (Meyer-Bernstein and Morin, 1999). For experiment 2, only animals with cannula tips localized in the SCN were used for analysis in the post lesion/vehicle conditions. Typical results of 5,7-DHT infusions into the SCN for both experiments resulted in nearly complete 5-HT fiber loss in the SCN while vehicle injection spared 5-HT fibers (Figure 3.2). Only those animals receiving complete, or near complete lesions of the serotonin fibers in the SCN were used in the analysis.
3.6 Experiment 2: Within Subjects Design
3.6.1 Control Stimulation Procedures
Control data was taken from experiment 2 where the animals were connected to the stimulator apparatus for 2 hours from CT4-6 but no current was passed. Those animals receiving the control procedure first (n=6) had an average phase advance of 0.13
±0.16 hrs with 2 animals showing slight phase delays and the rest showing slight phase advances. Those animals receiving the control procedure as the second manipulation 52
(n=3) showed an average phase advance of 0.02 ±0.12 hrs. In this case, only one animal showed a minor phase delay. Control data was not collected for two animals in this case as they did not survive the electrical stimulation procedure and one animal lost a headcap.
For the control animals, all of the data was used regardless of correct electrode placement in the MRN. Since there was no main effect of order of manipulation (F(iji8)=0.05, p>0.05) the control data was pooled together (Figure 3.3). Overall then, the control manipulation procedure resulted in an average phase shift of 0.09 ± 0.11 hrs. This confirmed that the control procedure has no appreciable effect on the clock, regardless of if the animal had been connected to the stimulator before or not. A representative actogram showing a control manipulation procedure is displayed (Figure 3.4 A).
3.6.2 Stimulation of the Median Raphe
Only those animals showing having an electrode tip located in the raphe were included in the analysis. Those animals receiving a stimulation of the MRN for the first manipulation (n=6) showed an average phase advance of 0.88 ± 0.28 hrs. Those animals receiving stimulation of the MRN as the second manipulation (n=3) showed an average phase advance of 0.8 ± 0.25 hrs. Again, since there was no order effect, data was summed together. Overall then, stimulation of the MRN produced an average phase shift of 0.85 ±
0.19 hrs. When only data for animals in both the control and stimulation groups (n=5) was used for analysis, a dependent group t-test revealed a significant difference between control and stimulation procedures (t(4)=4.49, p<0.05). An actogram displaying a phase advance to MRN stimulation is displayed (Figure 3.4 B). In general, it seemed that the closer the electrode tip was situated to the MRN the larger the resulting phase shift was. 53
Following microinjection of either vehicle, or 5,7-DHT, animals were once again allowed to establish a free running rhythm in DD before being stimulated again. Only those animals with electrode tips confirmed to be in the MRN region as well as cannulas confirmed to be in the SCN region were included in the analysis. Those animals receiving a successful injection of vehicle into the SCN (n=2) showed an average phase advance of
0.98 ±0.13 hrs to subsequent stimulation of the MRN (Figure 3.5). A dependent group t- test (n=2) revealed no significant difference between pre-sham lesion MRN stimulation and post-sham lesion injection phase shifts (t(i)=1.76, p=0.33). Those animals receiving successful lesion of the serotonergic input in the SCN (n=2) showed an average phase advance of 0.04 ± 0.02 hrs (Figure 3.5). A dependent group t-test also revealed no significant difference between pre-lesion and post-lesion phase shifts (t(i)=1.59, p=0.35).
The absence of a significant effect in this case was likely due to lack of power. Two of the animals in the lesion group died following stimulation and one animal in the vehicle group suffered from an infection under the headcap and was euthanized.
3.7 Experiment 2B: Between Subjects Design
3.7.1 Stimulation of the Median Raphe
Given the problems inherent in experiment 2 and low final sample size, we conducted a between subjects design to minimize time of electrode implant before stimulation, the total amount of stimulation, the age of the animal and total time in DD.
Those animals receiving injection of vehicle into the SCN with electrode tips localized in the MRN (n=4) showed an average phase advance of 0.82 ± 0.27 hrs to electrical 54 stimulation (Figure 3.6). Those animals receiving a successful lesion in the SCN (n=4) showed an average phase delay of-0.24 ± 0.16 hrs (Figure 3.6). This phase delay was largely due to one of the animals showing a delay of-0.72 hrs to the stimulation. The other lesioned animals showed smaller delays of less than -0.17 hrs or very little shift at all. The actogram of an animal that received a serotonergic lesion of the SCN and subsequent stimulation of the MRN is displayed (Figure 3.7). An independent group t-test revealed a significant difference between the lesion and sham lesion group phase shifts
(t(6)=3.41,p<0.05).
3.8 Discussion
3.8.1 What is the Zeitgeber?
In these experiments we attempted to determine if the non-serotonergic projections from the MRN to the SCN were important for non-photic phase resetting. If so, this may have indicated a role for cells containing VGLUT3 in bringing about these shifts. Although our findings were troubled by low sample sizes due to high initial mortality in the lesion group, in general, we found that midday 2 hour long stimulations of the MRN in DD across both studies resulted in average phase advances of 0.89 ±0.15 hrs. Control stimulation procedures where the animal was connected to the stimulation apparatus but no current was passed did not appear to be the cause of these shifts as on average, these procedures only resulted in phase advances of about 0.09 ± 0.11 hrs. There also did not appear to be an order effect as to whether the animal had been connected to the stimulation apparatus prior to stimulation or not. Following a successful lesion of the 55 serotonergic input into the SCN, electrical stimulation of the raphe produced very little effect on circadian phase, sometimes resulting in small phase delays. Across both studies,
SCN serotonin lesioned animals displayed an average phase delay -0.15 ± 0.12 hrs. This indicates that serotonergic projections are important for midday phase advances. Since there has been reported to be a very high degree of co-localization of 5-HT with
VGLUT3 (Mintz and Scott, 2006), it cannot be ruled out that VGLUT3 is the important factor in these shifts, however.
The nature and difficulties encountered with the MRN stimulation experiments bring several questions to light. The first problem is whether or not it is a real phenomenon, or confounded by the fact that there is damage to the MRN cells following stimulation. A study involving bilateral lateral ventricle infusions serotonin neurotoxin
5,7-DHT resulting in near complete widespread serotonin cell loss found immediate advanced activity onsets and delayed activity offsets resulting from a substantial expansion of the active period (Smale et al., 1990; Morin and Blanchard, 1991).
Neurotoxin specific lesions of the MRN 5-HT cells resulted in an immediate large advance in activity onset but had no effect on offset (Meyer-Bernstein et al., 1997). In our study, there was no noticeable expansion of the active phase following stimulation of the median raphe. In some cases the active period appeared to be attenuated. This indicates that the phase advances seen following MRN stimulation are not likely to be due to cell death at the simulation site. In several instances there was also the appearance of transients, or successive shifts of activity onset as the clock adjusted to the new time indicating, again, that activation of MRN projections to the SCN was involved in these shifts. 56
Another question arising is what exactly about the nature of MRN stimulation is resulting in the non-photic phase shift. It could be the case that stimulation of the MRN is causing excessive motor activity or a midday sleep deprivation that is resulting in the phase advance and it is not directly due to the MRN to SCN projection. It has been hypothesized that the positive or negative emotional valence of a stimulus may itself be the zeitgeber. For example, access to a novel running wheel has been used as a reward for hamsters in order to produce a conditioned place preference (Ralph et al., 2002).
Equivalent non-photic phase shifts were found when comparing rewarding brain stimulation to footshocks at various circadian times (Cain et al., 2004). This implies that an important factor for a non-photic zeitgeber is some sort of attached significance, be it positive or negative. MRN stimulation might have enough of an associated noxiousness to be a true non-photic stimulus. Stimuli judged to be stressful have been surprisingly unsuccessful at shifting the clock. A study using 3 hrs of sleep deprivation by gentle handling found significant potentiation of midday phase advances with prior administration of a Cortisol inhibitor. Sleep deprivations by gentle handling resulted in phase shifts of 107 ± 16 min that were potentiated to 149 ± 12 min by Cortisol inhibition
(Mistlberger et al., 2003). It is entirely possible then, that MRN stimulation is resulting in a sleep deprivation that is shifting the clock, but the shifts are attenuated due to the stressful nature of the stimulation. The alternative is that since our electrical stimulations were only 2 hrs, arousal was not sustained as long as the 3 hr sleep deprivations to induce the greatest phase resetting response. If this were the case, it would be expected that all animals would have shown a greater response to stimulation; however phase shifts to
MRN stimulation varied anywhere from 20 min to close to 2 hrs. The alternative 57 explanation for the varying sizes of phase shifts is the distance from the MRN that the electrode tips were situated. An interesting follow up study might use Cortisol inhibition coupled with MRN stimulation to determine if shifts could be potentiated.
3.8.2 Circling Behavior
One issue that needs to be addressed is the behavior apparent during the stimulation. Often during the stimulation procedure, animals would begin running in leftward circles, first turning their heads and then using their forelimbs to turn their bodies such that they appeared to be chasing their own tails. Similar behaviors in rats were found when stimulating midbrain and brainstem midline structures that elicited a behavioral circling phenomenon (Yeomans and Linney, 1985). It was found that axons eliciting these circling behaviors could be activated at 200 uA up to 1 mm away from electrode tip at parameters similar to our study (Yeomans et al., 1986). Thus, even with an exact electrode placement in the MRN nucleus, low threshold axons mediating circling behavior would be recruited by the stimulation. Through further study, it was surmised that the superior colliculus and rostral medial tegmentum projected through the pons to elicit body movements (Tehovnik and Yeomans, 1986). It is possible that in this study, more or less bilateral activation of these pathways resulted in those animals that appeared to be highly aroused, but did not circle. In order to approximate the radius of the field of stimulation field, the formula R=Vl/K where R is the radius of the stimulation field, I is the stimulation current in microamps and K is the current distance constant is usually used (Ranck, 1975). This formula was used in a similar study of midbrain pons stimulation in a site near where monopolar stimulations took place (Buckenham and 58
Yeomans, 1993). Even determining whether cells or fibers become activated, and to what distance from the electrode tip this occurs in the monopolar case is an extremely complex proposition (Ranck, 1975). Therefore, it cannot be determined to any sort of accurate measure how much of the bipolar stimulation we delivered would activate the surrounding areas. Based on histology of electrode placement and the very common occurrence of leftward circling, however, we can assume that our stimulations activated areas outside of the MRN such as the tectospinal tract or ventromedial tegmentum.
Electrode tips localized outside of the MRN and paramedian raphe, usually directly in these areas rarely resulted in any sort of appreciable shift.
It might be argued that the motor activity elicited from this circling behavior was what resulted in the non-photic phase shift. It was determined that the amount of overall activity in a novel wheel access manipulation determined the extent of a phase shift, with animals running greater than 4000 wheel revolutions (2.5-5 km) in 3 hrs showing the greatest shifts (Janik and Mrosovsky, 1993). Animals running very little during the manipulation showed relatively small shifts. In the sleep deprivation studies, it was estimated that as little as 0.08 km is travelled in 3 hrs (Antle and Mistlberger, 2000).
In this study only a very rough estimate of total distance travelled could be taken.
Since our hamsters were around 13 cm long, then the total circumference would be the rough estimate of distance travelled when engaging in circling behavior. It was estimated that the animal would travel approximately 40 cm per circle. Since it seems to take a distance of around 2.5 km or 250000 cm in order to produce a phase shift from motor activity, it would take the animal approximately 6250 revolutions in 2 hrs. This means the animal would have to make at least 52 rotations per minute. Since no animal studied 59 continued to circle for the entire 2 hr manipulation, most only circled for 1 hr and personal observations of the circling behavior found that it occurred at a speed lower than
1 revolution per second, it is highly unlikely that the total distance travelled would be anywhere close to that needed for an appreciable shift from motor activity. The total distance travelled during the stimulation was definitely greater than the 0.08 km found in sleep deprivations though. In pilot work and throughout the experiments, there were 4 animals that showed strong circling behavior but did not show phase shifts. These 4 animals only showed an average phase shift 12 + 2 min as further evidence against motor activity causing the phase shifts. The histology of these animals also showed that electrode tips were located outside the MRN in the tectospinal tract, pons or tegmentum.
3.9 Experimental Design Issues
The original conception of the experimental design for electrical stimulation of the raphe proved unwieldy. As the total stimulation time was 2 hrs, it was necessary for activity onsets to be more than 2 hrs apart in order to perform the stimulation on multiple animals per day. Unfortunately, onsets fell relatively close to one another and it thus it took 10-12 days to accomplish one round of manipulations as well as an additional 10 days to collect post manipulation data on the last animal. With cage changes only able to occur in between manipulations, it took an additional month of so to complete both stimulations and control stimulation data on all 12 animals. Following this lengthy period of time, lesion/vehicle injections took place before a second stimulation was applied to the animals after they resumed free running. This experimental design proved difficult due to the length of time of having chronic electrode implants. This long time may have 60 allowed for conditions within the brain surrounding the electrode to change. The time of placement also allowed for minor infections around the cannula site, or within the skull to foster causing some headcaps to loosen and fall off. Through personal communication with Dr. J.D. Glass (Kent State, Ohio), it was determined that repeated measures stimulation of the dorsal raphe was often not successful (Glass et al., 2003). This could be due to the fact that there was cell death in the area of stimulation following such a prolonged activation. It is possible that gliosis around the electrode placement would have occurred that might have altered the resistance at simulation site. It might be possible to determine if this was a major issue by staining for glial fibrillary acidic protein as a marker of glia at the electrode implant site.
Finally, the issue of the considerable mortality in the lesion group was puzzling.
There did not appear to be any significant immediately visible excess damage at the electrode tip sites. In some cases of neurotoxin administration, there was likely some ventricular leakage as there was 5-HT cell loss in the DRN. In some cases, it appeared that electrode placements in those animals that survived stimulation were located in the more rostral or caudal aspects of the MRN, however this was not always the case.
Although it was not evaluated, cell loss in the MRN appeared to be minimal. Even if there was cell loss, the projection from the MRN to the SCN is very small (see above).
Due to the sensitive area in which the MRN is situated, loss of cells in the pons or tegmentum could also have led to death. What likely occurred is that the initial concentration we used for the 5,7-DHT was too high for our experimental purposes.
There was significant mortality in the animals receiving infusions of the freshly mixed neurotoxin. With repeated use of the 5,7-DHT aliquots, however, this probably allowed 61 time for some of the neurotoxin to oxidize. Animals receiving doses of the older neurotoxin still showed substantial loss of serotonin fibers in the SCN however had a
100% survival rate following stimulation of the MRN.
In sum then midday phase advances to electrical stimulation of the MRN appear to be dependent on the serotonergic input into the SCN. Due to the potential stressful nature of these stimulations, and/or the duration, these phase advances may not have been the maximum possible shifts. A follow-up study using a Cortisol blocker or longer duration (3 hr) stimulations to more closely approximate non-photic stimuli would be necessary to clear up these issues. The zeitgeber in this case may have been sleep deprivation due to the sustained arousal throughout the experiment, but it was likely not due to the increased motor activity induced by the stimulation.
Chapter Four: General Discussion
4.1 Conclusions
The raphe projection to the SCN has been implicated in non-photic phase shifts.
We wanted to determine whether release of 5-HT, or another neurotransmitter from this raphe projection was the mechanism by which these phase shifts occur in the SCN. Since non-serotonergic cells in the raphe are not well characterized, we first sought to describe some of these non-serotonergic projections. Through the use of retrograde tract tracing, we found that some of the non-serotonergic cells in the raphe contain VGLUT3 and project to the SCN. VGLUT3 has been hypothesized to be involved in a novel form of glutamate signaling where glutamate is stored and released from the cell body. The 62 presence of VGLUT3 in terminals as well suggests that it releases glutamate there as well. Since we also found VGLUT3 was co-expressed in a subset of serotonergic cells, this raised the possibility that some cells in the raphe co-transmit two or more neurotransmitters. We also found a subset of cells in the MRN that were single labelled for the tracer indicating a yet unknown neurotransmitter input into the SCN.
In order to determine if the serotonin input into the SCN was necessary for non- photic phase shifts we next conducted a study using electrical stimulation of the raphe.
Midday electrical stimulation of the MRN was conducted in SCN sham, or 5-HT lesioned animals. Electrical activation the raphe in sham lesioned animals was predicted to result in a phase advance of the hamster circadian rhythm of wheel running. Electrical activation of just the non-serotonergic projections to the SCN in the 5-HT lesioned animals would determine if 5-HT was necessary to shift the clock. If it were the case that activation of only the non-serotonergic projections in the lesioned animals was sufficient to phase advance the circadian clock, raphe cells containing VGLUT3 might be important, or responsible for these phase shifts. Sham lesioned animals with intact serotonin input into the SCN showed a phase advance to midday electrical stimulation of the raphe. SCN 5-HT lesioned animals failed to phase advance to electrical stimulation of the raphe and often showed small phase delays. This indicates the importance of the 5-
HT input into the SCN for non-photic phase shifting although secondary loss of other serotonin terminals and cells could not be evaluated. A complex picture of raphe signaling is beginning to emerge. The precise role of VGLUT3 still needs to be determined along with the role of the rest of the non-serotonergic raphe projections to the
SCN. Since 5-HT in the SCN has already been implicated in modulating the response to 63 light and now in the induction of midday phase advances, the other non-serotonergic
MRN projections may play a modulatory role in these processes.
4.2 Future Directions
An interesting, but extremely difficult and time consuming undertaking would be to attempt core or shell specific SCN iontophoresis of a retrograde tract tracer. This would allow us to elucidate what may be some very specific underlying connections that have been previously overlooked. This approach could be coupled with an anterograde tracer approach as well. For example, if further core iontophoresis reveals a potential ventromedial DRN projection to the SCN, then application of anterograde tracer could be done in the DRN to confirm this finding. Tract tracing studies report that retrograde or anterograde tract tracing has taken place from a structure eg. the SCN, but fail to take into account the heterogeneity of that structure.
It will also be necessary to clarify the role of VGLUT3. The evidence above suggests that VGLUT3 is unlike VGLUT1 and VGLUT2 as it also concentrates glutamate for release from the cell body as well as the terminals. It should be determined to what extent the presence of VGLUT3 in the cell body determines whether or not that particular cell releases glutamate from the terminals.
In these experiments, we found that serotonergic raphe projections are important for non-photic phase shifting. If this is true, then a serotonin antagonist into the SCN should block phase shifting to raphe stimulation. Since a substantial proportion of 5-HT appears to colocalize with VGLUT3, it also might be the case that a glutamate antagonist will be needed to block MRN induced phase shifts. Understanding how the raphe system 64 communicates will be an important step in neuroscience research as it projects widely to most other areas of the brain and is involved in the modulation of many other functions outside of circadian rhythms.
4.3 Clinical Importance/ Relevance
An important question in the study of non-photic phase shifting could be whether or not this same phenomenon exists in humans. It has already been well established that mating cues, opportunities to hoard, and nesting behavior can be important zeitgebers for some of the common lab animals (Mrosovsky, 1996). There is evidence that mealtime and scheduled exercise can have modest effects on the human circadian pacemaker, although these are very weak zeitgebers and findings may be confounded by study lighting conditions (reviewed by Mistlberger and Skene, 2005). Social factors do not appear to play a major role in resetting the human pacemaker. Although since social factors are extremely important in human society, if they were powerful zeitgebers then they might do more harm than good by constantly setting and resetting the clock. Since
Syrian hamsters appear to be solitary creatures and only rarely engage in social contact
(Gattermann et al., 2008), it stands to reason that these non-photic cues would be of much greater importance for entrainment of behavior such things as mating.
One study looking at changes in melatonin secretion in humans as a marker of circadian phase showed a night or morning scheduled bout of hour long exercise produced phase delays of about an hour whereas afternoon and evening exercise produced smaller phase advances (Buxton et al., 2003). Experiments using human volunteers seem to preclude the possibility of using longer exercise bouts as a 65 manipulation as forcing a volunteer to exercise for up to 3 hrs might be difficult. Another study using very dim light to control for the possible lighting confounds found that only nighttime scheduled exercise was capable of producing phase delays of less than an hour
(Barger et al., 2004). Light is by far the most powerful factor in human circadian phase resetting, and therefore has been a potential confound in many of the human non-photic shifting studies so far (Mistlberger and Skene, 2005).
The human circadian pacemaker runs at a period very close to 24 hours (-24.2 h) making the need for photic and non-photic phase shifts to be very small in order to remain entrained (Burgess and Eastman, 2008). This might make any phase shift studied in humans possibly too small to detect. Furthermore, the conditions under which the study of any non-photic stimulus would have take place in very dim light, or darkness that would be unrealistic for longer studies using human volunteers (Barger et al., 2004).
Given these difficulties in studying non-photic stimuli in humans, a unique opportunity arises when studying blind populations. If non-photic stimuli are important zeitgebers, it would be expected that blind subjects would rely heavily on them in order to remain entrained to a 24 hour day. In an outpatient study of blind individuals lacking a conscious perception of light, and incapable of entrainment to light, 9 out of 15 participants remained synchronized to the 24 hr day (Klerman et al., 1998). The non-photic stimuli resetting the clock in this case were not elucidated, but it was assumed they would have a small phase advancing effect in order to keep these individuals entrained to a 24 hr day.
This suggests that there are individual differences in the response to non-photic stimuli as some individuals were capable of responding to them and others were not. 66
The importance of continuing to study non-photic phase shifting in an animal model may have some potential benefits for blind humans if it can help to entrain rhythms. Exploring the effects of scheduled exercise can have potential benefits for adolescents as well. It is a well-established phenomenon that teenagers exhibit a phase delayed sleep pattern where they tend to fall asleep later in the evening and sleep in later in the morning. Since many adolescents are involved in organized sports, it should be established whether the timing of these activities would exacerbate or improve the altered sleep patterns (Mistlberger and Skene, 2005). Since animal studies involving non-photic stimuli have shown the potential for shifting circadian rhythms by several hours, it also hints at a mechanism by which we can do the same in humans. Discovering how these stimuli work in humans and the development of pharmacological tools to mimic those effects could have tremendous applications in assisting trans-meridian travelers and aiding shift workers.
Another fascinating avenue of investigation involves the human depression literature. It has been known for sometime that there are altered circadian rhythms in patients suffering from mental disorders (eg. Elithora et al., 1966). The circadian rhythm of serotonin expression is altered and overall levels of serotonin are also reduced in patients suffering from depressive illnesses (Pietraszek et al., 1992). Thus, increasing the availability of serotonin through the use of selective serotonin reuptake inhibitors has proven effective in combating depressive symptoms (Kernick, 1997). Through the non- photic literature as well as human studies, it has been established that during depression there is reduced physical activity and that exercise can increase serotonin release and have a positive effect on mood (Young, 2007; Salmon, 2001). A complex model has been 67 devised suggesting a relationship between serotonin, depression, the circadian clock, activity and sleep (Mistlberger et al., 2000). This model incorporates two major non- photic stimuli: sleep deprivation and physical activity. These stimuli have known effects on the circadian system. Since these two stimuli have also been shown to have antidepressant effects, it can be asked whether serotonin is acting at the clock in order to reduce the symptoms of depression through circadian phase resetting (Mistlberger et al.,
2000). In these studies, we found evidence that serotonin release in the SCN is important for circadian phase resetting. This may indicate that future pharmacological interventions for depressive illness could focus on the circadian clock. 68
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Figure 2.1 Photomicrograph cases of iontophoresis into the SCN
Figure 2.1: A-E: The suprachiasmatic nuclei of 5 animals that received successful iontophoresis of the retrograde tract tracer cholera
toxin beta subunit. The cholera toxin was labelled using immunocytochemistry and visualized with a Cy2 conjugated fluorescent
secondary antibody. The darker stained areas on each section are where the iontophoresis tip was localized. Scale bar: 200 urn. 95
Figure 2.2 Representative triple label histology and mean distribution of cell types retrogradely labelled in the MRN
MRN
30.0% 28.0% A 21i%
Tot CTb 5-HT VGLUT3 5-HT+ VGLUT3 Cell Type
Figure 2.2: A: section of triple labelled median raphe showing CTb immunoreactive cells.
Red indicates the presence of vesicular glutamate transporter 3, blue indicates cells
that contain serotonin, and green are cells that stained for cholera toxin. Arrows
indicate cells containing the cholera toxin that were analyzed for the presence of
VGLUT3 and 5-HT. B: The average (rounded) proportions of each cell type found to
project to the SCN following successful retrograde tracing. The means are displayed
± standard error. Counts were averaged from twelve alternate 35um MRN sections
from each animal (n=5). 96
Figure 2.3: High magnification representative examples of each cell type found to project
to the SCN. Each row as designated by a letter represents a single case and each
column displays a label for that case. A: a single label CTb cell. Staining indicated a
visible cell body in the cholera toxin channel, but this was not apparent in the 5-HT,
or VGLUT3 channels. B: A cell double labelling for CTb and 5-HT, but not
VGLUT3. C: A cell double labelling for CTb and VGLUT3, but not 5-HT. D: A cell
triple labelling for CTb, 5-HT and VGLUT3. 97
Figure 2.3 Photomicrographs displaying representative examples of retrogradely labelled cell types in the MRN 98
Figure 2.4 Iontophoresis site in the SCN and retrograde labelling in the DRN for one case
Figure 2.4: A: Another section for Figure 2.4 A is displayed in Figure 2.1 A.
Ionotophoresis in this case resulted in unilateral staining of the DRN as displayed in
B. Scale bars in the SCN sections are 200um. Scale bars in the DRN sections are
lOOum. Figure 2.5 Mean distribution of cell types retrogradely labelling in the DRN 501 19P% DRN
40H
30H o o 36.7% = 20- o 25.9% 22.4% 10 15.0il% Tot CTb 5-H VGLUT3 5-HT+ VGLUT3 Cell Type
Figure 2.5: Mean number of cells staining for cholera toxin found in the DRN of those
animals receiving successful iontophoresis into the SCN. The bar labelled CTb
indicates single labelled cholera toxin, the bar labelled 5-HT were double labelled for
both CTb and 5-HT etc. Means are displayed ± standard error. 100
Figure 3.1: Photomicrographs of electrode tip placement histology in the MRN. A: An
animal from experiment 2 with an electrode tip localized rostrally and dorsolateral^
within the paramedian raphe. B: An animal from experiment 2B with an electrode tip
localized directly in the MRN. The electrode track is larger in this case as the
electrode tips were separated slightly before implantation. DRN: dorsal raphe
nucleus, xscp: decussation of the superior cerebral peduncle. Scale bar 400 urn. 101
Figure 3.1 5-HT labelled histology showing MRN electrode tip placements 102
Figure 3.2 5-HT labelled sections showing intact and lesioned serotonergic input into the SCN
Figure 3.2: 5-HT labelled DAB stained SCN sections. A: displays a representative
example of an animal treated with vehicle (0.5% ascorbic acid in sterile saline). The
dark stained fibres show a dense serotonergic input into the ventromedial SCN. B:
displays an SCN section of an animal treated with 5,7-DHT. Dark arrows show a near
complete loss of serotonergic input into the SCN. The remaining fibers in this case
appeared to be unhealthy. 3V: Third ventricle, OX: Optic chiasm. Scale bar is 100
um. 103
Figure 3.3 Mean phase advances to control and MRN stimulation for experiment 2
#\ 1.0J in 0.84
C/D 0.6-
CD .c 0.4-^
0.2H
0.0 Control Stimulation
Figure 3.3: Average phase advances in hours to midday control and stimulation of the
MRN (n=8 per group) for experiment 2. Data was summed across counterbalanced
conditions where one group received a control and then a stimulation procedure and
the other group received stimulation and then the control procedure. Control and
stimulation procedures took place at CT4 in each animal's activity rhythm and lasted
2 hrs. Only animals with data collected from both conditions were used in the
statistical analysis. Means are displayed ± standard error. 104
Figure 3.4: Actograms displaying a control manipulation procedure and stimulation of the
MRN. Hours are displayed on the horizontal axis, and days are displayed vertically.
Wheel counts were summed into 10 minute bins and displayed by dark vertical
deflections on the horizontal lines. A: an animal that was connected to the stimulation
apparatus but no current was passed. Calculated activity onsets are displayed as the
red dots. The regression lines were fitted to these activity onsets in order to calculate
phase shifts. B: displays an animal that received electrical stimulation of the MRN.
The time of stimulation is indicated by the red box with the yellow lightning bolt. The
phase shift was the difference between the two regression lines on the manipulation
day. Figure 3.4 Actograms displaying an animal that received the control manipulation and another that received stimulation of the MRN
I 1
,_ • TTT D r*"' 1 Tpr
_ • j M I——--j'-TH * i. . A . it .. _^J 1 1"*" 1 T"|". ^T MRN DEB
stimulation • •• * * Q •g 1 f"Tl;PH| M JTTTFP • r^n T"T-i irrrr^ m M ''TTF ^•"»
d 1 Til ^ T 1 M !l • P 1 -i^n ^ rr»i m 106
Figure 3.5 Mean phase advances to MRN stimulation following cannula injection of vehicle, or 5,7-DHT B'l 1.0 J C/) ^,0.8
CO 0.6 0.21 0.0 Sham 5,7-DHT Figure 3.5: Average phase advances in hours to midday stimulation of the MRN following injection of either vehicle (n=2) or 5,7-DHT (n=2) into the SCN in experiment 2. Stimulations took place from CT4-6. Means are displayed ± standard error. Figure 3.6 Mean phase shifts to MRN stimulation following vehicle of 5,7-DHT injection for experiment 2B 1.2 1.0. 0.8. 'E 0.6. *r°"4" g02. £ o.o. 5,7-DHT (0 Sham XI-0.2- Q. -0.4- -0.6- .nft. Figure 3.6: Average phase shifts in hours to midday stimulation of the MRN following injection of either vehicle (n=4) or 5,7-DHT (n=4) into the SCN in experiment 2B. Stimulations took place from CT4-6. Phase advances are displayed as positive numbers, whereas the phase delays are displayed as negative. Means are displayed ± standard error. 108 Figure 3.7 Actogram displaying an animal that received a complete lesion of the serotonergic input into the SCN and subsequent stimulation of the MRN Figure 3.7: Actogram showing the activity record of an animal who received a complete lesion of the serotonergic input into the SCN and subsequent stimulation of the MRN. Hours are displayed on the horizontal axis, and days are displayed vertically. Wheel counts were summed into 10 minute bins and displayed dark vertical deflections on the horizontal lines. The solid red box with the lightning bolt indicates the time when the animal was stimulated (8 hrs before activity onset for two hours).